U.S. patent number 10,092,600 [Application Number 15/388,428] was granted by the patent office on 2018-10-09 for method of preparing an adipose tissue derived matrix.
This patent grant is currently assigned to Musculoskeletal Transplant Foundation. The grantee listed for this patent is MUSCULOSKELETAL TRANSPLANT FOUNDATION. Invention is credited to Bryan Choi, Yen-Chen Huang, Asia Ivery, Manh-Dan Ngo, Benjamin Schilling.
United States Patent |
10,092,600 |
Huang , et al. |
October 9, 2018 |
Method of preparing an adipose tissue derived matrix
Abstract
An acellular soft tissue-derived matrix includes a collagenous
tissue that has been delipidated and decellularized. Adipose tissue
is among the soft tissues suitable for manufacturing an acellular
soft tissue-derived matrix. Exogenous tissuegenic cells and other
biologically-active factors may be added to the acellular matrix.
The acellular matrix may be provided as particles, a slurry, a
paste, a gel, or in some other form. The acellular matrix may be
provided as a three-dimensional scaffold that has been
reconstituted from particles of the three-dimensional tissue. The
three-dimensional scaffold may have the shape of an anatomical
feature and serve as a template for tissue repair or replacement. A
method of making an acellular soft tissue-derived matrix includes
steps of removing lipid from the soft tissue by solvent extraction
and chemical decellularization of the soft tissue.
Inventors: |
Huang; Yen-Chen (East
Brunswick, NJ), Ivery; Asia (Hammonds Plains, CA),
Choi; Bryan (San Marcos, CA), Schilling; Benjamin
(Pittsburgh, PA), Ngo; Manh-Dan (Matawan, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
MUSCULOSKELETAL TRANSPLANT FOUNDATION |
Edison |
NJ |
US |
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Assignee: |
Musculoskeletal Transplant
Foundation (Edison, NJ)
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Family
ID: |
51352835 |
Appl.
No.: |
15/388,428 |
Filed: |
December 22, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170119826 A1 |
May 4, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14446629 |
Jul 30, 2014 |
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61860043 |
Jul 30, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
35/35 (20130101); A61L 27/3683 (20130101); A61L
27/3604 (20130101); A61L 2430/40 (20130101) |
Current International
Class: |
A61K
35/35 (20150101); A61L 27/36 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
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|
|
|
0518389 |
|
Dec 1992 |
|
EP |
|
2007037572 |
|
Apr 2007 |
|
WO |
|
2008154623 |
|
Dec 2008 |
|
WO |
|
2009102452 |
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Aug 2009 |
|
WO |
|
2011019822 |
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Feb 2011 |
|
WO |
|
2011087743 |
|
Jul 2011 |
|
WO |
|
2012002986 |
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Jan 2012 |
|
WO |
|
2012166784 |
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Dec 2012 |
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WO |
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2014052376 |
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Apr 2014 |
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WO |
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2015017500 |
|
Feb 2015 |
|
WO |
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Other References
Wang, L. et al. Combining Decellularized Human Adipose Tissue
Extracellular Matrix and Adipose Derived Stem Cells for Adipose
Tissue Engineering. Acta Biomaterialia 9:8921-31, Jun. 2013. (Year:
2013). cited by examiner .
Poon C. et al. Preparation of an Adipogenic Hydrogel from
Subcutaneous Adipose Tissue. Acta Biomaterialia 9(3)5609-5620, Mar.
2013. (Year: 2013). cited by examiner .
Australian Patent Examination Report No. 1 regarding Australian
Patent Application No. 2014296259, dated Jun. 2, 2016. cited by
applicant .
Colombian Office Action regarding Colombian Patent Application No.
16-049.384, dated Mar. 22, 2016. cited by applicant .
International Search Report and Written Opinion for International
(PCT) Application No. PCT/US2014/025619, dated Jun. 30, 2014. cited
by applicant .
International Search Report and Written Opinion of the
International Searching Authority in regard to International
Application No. PCT/US2014/048797, dated Oct. 9, 2014. cited by
applicant .
Non-Final Office Action dated Sep. 22, 2016 by the USPTO regarding
U.S. Appl. No. 14/446,629. cited by applicant .
Choi, J. et al., Human extracellular matrix (ECM) powders for
injectable cell delivery and adipose tissue engineering, J Control
Release, 139, 1, (2009), p. 2-7. cited by applicant .
Clarke, KM, et al., Intestine submucosa and Polypropylene Mesh for
Abdominal Wall Repair in Dogs, Journal of Surgical Research, vol.
60, Iss. 1, pp. 107-114, (Jan. 1996). cited by applicant .
Coleman III, W. et al., Autologous Collagen? Lipocytic Dermal
Augmentation A Histopathologic Study, J. Dermatol. Surg. Oneal.,
vol. 19, pp. 1032-1040, (1993). cited by applicant .
Erdag, et al., Fibroblasts Improve Performance of Cul Tu Red
Composite Skin Substitutes on Athymic Mice, Burns, 30 (2004) pp.
322-328. cited by applicant .
Grauss, R.W. et al., Decellularization of Rat Aortic Valve
Allografts Reduces Leaftet Destruction and Extracellular Matrix
Remodeling, Journal Thoracic and Cardiovascular Surgery, 126,
2003-2010, (2003). cited by applicant .
Hara, A. et al., Lipid extraction of tissues with a low-toxicity
solvent, Analytical Biochemistry, 90, 1, (1978), p. 420-426. cited
by applicant .
Hubbell, J., Materials as morphogenic guides in tissue engineering,
Current Opinion in Biotechnology, 14, pp. 551-558, (2003). cited by
applicant .
Kropp, BP., et al., Experimental assessment of small intestinal
submucosa as a bladder wall substitute, Urology, vol. 46, Iss. 3,
pp. 396-400, (Sep. 1995). cited by applicant .
Kropp, BP., et al., Regenerative urinary bladder augmentation using
small intestinal submucosa: urodynamic and histopathologic
assessment in long-term canine bladder augmentations, J. Ural.,
vol. 155, Iss. 6, pp. 2098-2104 (Jun. 1996). cited by applicant
.
Oliver, et al., "Reconstruction of Full-Thickness Loss Skin Wounds
Using Skin Collagen Allografts", British Journal of Plastic
Surgery, 32 (1979), pp. 87-90. cited by applicant .
Prevel, CD., et al., Small intestinal submucosa: utilization for
repair of rodent abdominal wall defects, Ann. Plast. Surg., vol.
35, Iss. 4, pp. 374-380, (Oct. 1995). cited by applicant .
Schmidt, C. et al., Acellular vascular tissues: natural
biomaterials for tissue repair and tissue engineering,
Biomaterials, 21, (2000), p. 2215-2231. cited by applicant .
Sekiya, S. et al., Bioengineered cardiac cell sheet grafts have
intrinsic angiogenic potential, Biochemical and Biophysical
Research Communications, 341, pp. 573-582, (2006). cited by
applicant .
Takasaki, S. et al., Human type VI collagen: purification from
human subcutaneous fat tissue and an immunohistochemical study of
morphea and systemic sclerosis, J Dermatol, 22, 7, (Jul. 1995), p.
480-485. cited by applicant .
Ueda, Y. et al., Antigen clearing from porcine heart valves with
preservation of structural integrity, The International Journal of
Artificial Organs, vol. 29, No. 8, pp. 781-789, (2006). cited by
applicant .
Wang, L. et al., Combining decellularized human adipose tissue
extracellular matrix and adipose-derived stem cells for adipose
tissue engineering, Acta Biomater, 9, 11, (2013), p. 8921-8931.
cited by applicant .
Wellisz, T. et al., Ostene, a new water-soluble bone hemostasis
agent, J. Craniofac. Surg., vol. 17, Iss. 3, pp. 420-425, (May
2006). cited by applicant .
Wilshaw, S. et al., Production of an Acellular Amniotic Membrane
Matrix for Use in Tissue Engineering, Tissue Engineering, vol. 12,
No. 8, pp. 2117-2129, (2006). cited by applicant.
|
Primary Examiner: Gitomer; Ralph J
Attorney, Agent or Firm: Cole Schotz, P.C. Bodner; Marcella
M.
Parent Case Text
RELATED APPLICATIONS
The present application is a divisional of U.S. patent application
Ser. No. 14/446,629, filed on Jul. 30, 2014, now abandoned which
claims priority to U.S. Provisional Patent Application Ser. No.
61/860,043, filed Jul. 30, 2013, the disclosures of both of which
are incorporated by reference herein in their entireties.
Claims
We claim:
1. A method of preparing an adipose tissue-derived matrix,
comprising the steps of: (a) obtaining an adipose tissue; (b)
mechanically reducing the size of the adipose tissue; (c)
delipidating the adipose tissue by contacting the adipose tissue
with a polar organic solvent such that lipids are transferred from
the adipose tissue to the polar organic solvent, thereby producing
a delipidated adipose tissue from which substantially all of the
lipids native to the adipose tissue have been removed; (d)
decellularizing the delipidated adipose tissue after step (c),
thereby producing a delipidated, decellularized adipose tissue
which is essentially free of native cells and cellular
components.
2. The method claim 1, comprising the further step of separating
the adipose tissue from lipids released from the adipose tissue
during the mechanically reducing step (b).
3. The method of claim 1, wherein said step of mechanically
reducing the size of the adipose tissue includes grinding the
adipose tissue with a coarse grinding plate, then grinding the
ground adipose tissue with a fine grinding plate.
4. The method of claim 1, wherein said delipidating step includes
washing the adipose tissue with a polar organic solvent, wherein
the polar solvent includes a substituted hydrocarbon having a
number of carbon atoms in the range of two carbon atoms to six
carbon atoms, and said substituted hydrocarbon is selected from the
group consisting of a chlorinated hydrocarbon, a fluorinated
hydrocarbon, an alcohol, an ether, a ketone, an aldehyde, an ester,
an organic acid, and combinations thereof.
5. The method of claim 1, wherein said delipidating step includes
blending the adipose tissue with a polar organic solvent, Wherein
the polar solvent includes a substituted hydrocarbon having a
number of carbon atoms in the range of two carbon atoms to six
carbon atoms, and said substituted hydrocarbon is selected from the
group consisting of a chlorinated hydrocarbon, a fluorinated
hydrocarbon, an alcohol, an ether, a ketone, an aldehyde, an ester,
an organic acid, and combinations thereof.
6. The method of claim 1, wherein said delipidating step includes
homogenizing the adipose tissue with a polar organic solvent,
wherein the polar solvent includes a substituted hydrocarbon having
a number of carbon atoms in the range of two carbon atoms to six
carbon atoms, and said substituted hydrocarbon is selected from the
group consisting of a chlorinated hydrocarbon, a fluorinated
hydrocarbon, an alcohol, an ether, a ketone, an aldehyde, an ester,
an organic acid, and combinations thereof.
7. The method of claim 1, wherein said decellularizing step
includes contacting the delipidated adipose tissue with a detergent
solution, wherein said detergent is present in said solution at a
concentration in a range of from about 0.1% to about 5.0%
(w/v).
8. The method of claim 7, wherein the detergent solution includes a
deoxycholate salt.
9. The method of claim 7, wherein the detergent solution includes a
mixture of water and alcohol.
10. The method of claim 9, wherein the alcohol is present in the
detergent solution in an amount in a range of from about 20% to
about 40% by volume.
11. The method of claim 1, comprising the further step of soaking
the delipidated, decellularized adipose tissue in a disinfectant
solution.
12. The method of claim 11, comprising the further step of blending
the delipidated, decellularized adipose tissue with the
disinfectant solution.
13. The method of claim 11, wherein the disinfectant solution is an
acidic solution that includes peracetic acid.
14. The method of claim 11, wherein the disinfectant solution
includes an alcohol and a glycol.
15. The method of claim 11, wherein substantially all substances
that pose a significant risk of causing an immunogenic response in
a patient receiving said adipose tissue-derived matrix are removed
from the adipose tissue.
16. The method of claim 11, wherein substantially all native lipids
and native nucleic acids are removed from the adipose tissue.
17. The method of claim 1, comprising the further step of
contacting the delipidated, decellularized adipose tissue with a
solution having an acidic pH so as to form a gel.
18. The method of claim 1, comprising the further step of blending
the delipidated, decellularized adipose tissue in a polar liquid,
whereby the delipidated, decellularized adipose tissue and the
polar liquid form a flowable gel.
19. The method of claim 1, comprising the further steps of drying
the delipidated, decellularized adipose tissue, and reducing the
dried delipidated, decellularized adipose tissue to a dried
particulate form.
20. The method of claim 8, wherein the detergent solution includes
a mixture of water and alcohol.
21. The method of claim 12, wherein the disinfectant solution is an
acidic solution that includes peracetic acid.
22. The method of claim 12 wherein the disinfectant solution
includes an alcohol and a glycol.
23. The method of claim 12, wherein substantially all substances
that pose a significant risk of causing an immunogenic response in
a patient receiving said adipose tissue-derived matrix are removed
from the adipose tissue.
24. The method of claim 12, wherein substantially all native lipids
and native nucleic adds are removed from the adipose tissue.
Description
FIELD OF THE INVENTION
The present invention relates generally to matrices made from
decellularized soft tissues, including adipose, dermis, fascia,
muscle, pericardium, and other connective or membranous tissues,
and in particular, to such matrices as are suitable for
implantation into a living body in plastic surgery procedures,
including reconstructive or cosmetic surgery procedures.
BACKGROUND OF THE INVENTION
Techniques for restoring structure and function to damaged tissue
are used routinely in the field of plastic surgery, including
reconstructive and cosmetic surgery. Tissue transplantation is one
way of restoring structure and function by replacing or rebuilding
the damaged tissue. The transfer of biological material from one
individual to another can raise significant risks. One such risk is
tissue rejection, which can occur even in cases where there is a
good histocompatibility match. The risk of tissue rejection can be
reduced by processing tissues so that they become essentially free
of cell components (e.g., cellular membranes, nucleic acids,
lipids, and cytoplasmic components) that cause immunogenic
responses. Such tissues are sometimes referred to as
decellularized, cell-free, or acellular matrices. It is also
desirable to retain the growth factors that are required to promote
cellular ingrowth into the acellular matrix, eventually replacing
the acellular matrix with the patient's native tissue.
In particular, injectable soft tissue fillers have expanded the
non-surgical options available for volume replacement, facial
defect filling and rejuvenation in the aging face. Injectable soft
tissue fillers are widely used for both superficial and deep
aesthetic applications, including lip augmentation or rejuvenation
of the aging lip to restore shape and contour, fine line filling to
reduce the appearance of wrinkles around the eyes or mouth,
improvement of nasolabial folds, correction of eyelid deformities,
volume filling for the cheek and jawline, and for deep wrinkle or
scar filling.
There are many soft tissue fillers that are commercially available
for these applications, and in general, fall within the following
categories: autologous implant materials, collagens, hyaluronic
acid (HA), and biosynthetic polymers. Durability of these materials
varies from several weeks to years, or is considered permanent.
SUMMARY OF THE INVENTION
Embodiments of the present invention relate generally to
decellularized soft tissue-derived matrices (also referred to as
"acellular matrices") suitable for use in plastic surgery
procedures, including reconstructive and cosmetic procedures. The
matrices may be derived from a number of types of mammalian
tissues, including human tissues. Such types of tissues include,
but are not limited to, adipose, dermis, fascia, muscle,
pericardium, and other connective or membranous tissues. In
embodiments of the present invention, the matrices are in the form
of lyophilized (i.e., freeze-dried) particles. According to some
embodiments of the present invention, the particles are rehydrated
in a carrier for injection into a patient. According to some
embodiments of the present invention, the matrices are autologous
to the patient. In other embodiments, the tissues are allografts.
In yet other embodiments, the matrices are xenografts. According to
some embodiments of the present invention, the matrices are in the
form of sheets or strips. In other embodiments of the present
invention, the matrices are in a particulate form. In yet other
embodiments of the present invention, the matrices are in the form
of an injectable paste, gel, suspension, or slurry. According to
some embodiments of the present invention, the matrices are in a
lyophilized (i.e., freeze-dried) form. In other embodiments of the
present invention, the matrices are in a rehydrated form. According
to some embodiments, the matrices have been reseeded with cells.
According to some embodiments of the present invention, factors for
promoting and directing tissue ingrowth and differentiation (e.g.,
growth factors, such as VEGF or bFGF) have been added to the
matrices.
Other embodiments of the present invention relate to a process for
preparing acellular matrices from soft tissues. Some embodiments of
the process include the steps of: (1) isolating a desired soft
tissue obtained from a donor; (2) decellularizing the tissue by a
process which includes soaking the isolated tissue in a hypertonic
solution, soaking the isolated tissue in a surfactant, and soaking
or rinsing the processed tissue in sterile water; (3) disinfecting
the decellularized tissue; and (4) post-processing the tissue into
a desired form. According to some embodiments of the process, the
process includes a step of removing lipids from the isolated tissue
(i.e., delipidizing/delipidating the tissue), which may be
performed before decellularizing the isolated tissue. According to
some embodiments of the process, the delipidization step is
performed by agitating the isolated tissue in an alcohol.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be further explained with reference to
the attached drawings, wherein:
FIG. 1 is a schematic flowchart illustrating a process of preparing
an acellular soft tissue-derived matrix according to an embodiment
of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Detailed embodiments of the present invention are disclosed herein.
It should be understood that the disclosed embodiments are merely
illustrative of the invention that may be embodied in various
forms. In addition, each of the examples given in connection with
the various embodiments of the invention is intended to be
illustrative, and not restrictive.
The present invention relates generally to matrices made from
decellularized soft tissues, including, but not limited to,
adipose, dermis, fascia, muscle, pericardia, and other connective
or membranous tissues. Decellularized soft tissues, and methods of
making same, are also disclosed in the co-owned U.S. Pat. No.
7,723,108, issued May 25, 2010, which is incorporated herein by
reference. Acellular matrices of the present invention in a
particulate or slurry form may be used as bulking agents in
reconstructive or cosmetic surgery procedures (e.g., for filling
voids in tissue or smoothing wrinkles), and as scaffolds for tissue
repair and regeneration. In strip or sheet form, acellular matrices
according to the present invention may be used to provide
structural support to other tissues, and also to provide scaffolds
for tissue repair and regeneration. Examples of procedures in which
acellular matrices may be used include, but are not limited to,
volume replacement, facial defect filling and rejuvenation in the
aging face. Injectable acellular matrices may also be used for both
superficial and deep aesthetic applications, including lip
augmentation or rejuvenation of the aging lip to restore shape and
contour, fine line filling to reduce the appearance of wrinkles
around the eyes or mouth, improvement of nasolabial folds,
correction of eyelid deformities, volume filling for the cheek and
jawline, and for deep wrinkle or scar filling.
The term "soft tissues" refers generally to non-calcified tissues
from the mammalian body. For the purposes of the present
disclosure, such tissues include an adipose tissue, an amnion
tissue, an artery tissue, a cartilage tissue, a connective tissue,
a chorion tissue, a colon tissue, a non-calcified dental tissue, a
dermal tissue, a duodenal tissue, an endothelial tissue, an
epithelial tissue, a fascial tissue, a gastrointestinal tissue, a
gingival tissue, a growth plate tissue, an intervertebral disc
tissue, an intestinal mucosal tissue, an intestinal serosal tissue,
a ligament tissue, a liver tissue, a lung tissue, a mammary tissue,
a membranous tissue, a meniscal tissue, a muscle tissue, a nerve
tissue, an ovarian tissue, a parenchymal organ tissue, a
pericardial tissue, a periosteal tissue, a peritoneal tissue, a
placental tissue, a skin tissue, a spleen tissue, a stomach tissue,
a synovial tissue, a tendon tissue, a testes tissue, an umbilical
cord tissue, a urological tissue, a vascular tissue, a vein tissue,
and other non-calcified tissues.
Tissue Types
Tissues from which the acellular matrices of the present invention
may be formed, as well as tissues which may be augmented or
repaired using such acellular matrices are described more fully
hereinbelow.
1. Tissue Compartments
In multicellular organisms, cells that are specialized to perform
common functions are usually organized into cooperative assemblies
embedded in a complex network of secreted extracellular
macromolecules (i.e., the extracellular matrix (ECM)), to form
specialized tissue compartments. Individual cells in such tissue
compartments are in contact with ECM macromolecules. The ECM helps
hold the cells and compartments together and provides an organized
lattice or scaffold within which cells can migrate and interact
with one another. In many cases, cells in a compartment can be held
in place by direct cell-cell adhesions. In vertebrates, such
compartments may be of four major types, a connective tissue (CT)
compartment, an epithelial tissue (ET) compartment, a muscle tissue
(MT) compartment and a nervous tissue (NT) compartment, which are
derived from three embryonic germ layers: ectoderm, mesoderm and
endoderm. The NT and portions of the ET compartments are
differentiated from the ectoderm; the CT, MT and certain portions
of the ET compartments are derived from the mesoderm; and further
portions of the ET compartment are derived from the endoderm.
1.1. Extracellular Matrix
The ECM is an intricate network of secreted extracellular
macromolecules that largely fills the extracellular space in the
tissue compartments and comprises large polymeric complexes of
glycosaminoglycans (GAGs) and proteoglycans. GAGs are negatively
charged unbranched polysaccharide chains comprising repeating
disaccharide units. Each repeating disaccharide unit of a GAG chain
contains an amino sugar (e.g., N-acetyl glucosamine), which in most
cases is sulfated, and an -uronic acid (e.g., glucuronic or
iduronic acid). Four main types of GAG molecules are distinguished
based on sugar residues, type of linkage, number and location of
sulfate groups: (1) hyaluronan; (2) chondroitan sulfate and
dermatan sulfate; (3) heparan sulfate and heparin; and (4) keratin
sulfate.
GAG chains are inflexible and tend to adopt extended conformations
occupying a huge volume relative to their mass, forming gels even
at low concentrations. Their high density of negative charges
attracts cations, such as Na.sup.+, that are effective in osmotic
absorption of large amounts of water into the matrix. This creates
high turgor enabling the ECM to withstand compressive forces.
Hyaluronan (also termed hyaluronic acid or hyaluronate) (HA), which
comprises a regular repeating sequence of up to 25,000 nonsulfated
disaccharide units, serves many functions, many of which depend on
the binding of HA-binding proteins and proteoglycans, which are
either themselves constituents of the ECM or are integral
constituents of cell surfaces. For example, HA resists compressive
forces in joints as a major constituent of joint fluid serving as a
lubricant; serves as a space filler during embryonic development;
creates a cell-free space in the epithelial compartment to allow
cell migration during the formation of heart, cornea and other
organs; and plays a role in wound repair. Excess HA is usually
degraded by hyaluronidase.
All GAGs, except for HA, are covalently linked to proteins in the
form of proteoglycans. During their synthesis, the polypeptide
chain of proteoglycans is synthesized on membrane-bound ribosomes
and threaded into the lumen of endoplasmic reticulum, from which
they are sorted in the Golgi apparatus, and assembled with
polysaccharide chains. While still in the Golgi, proteoglycans
undergo a series of sequential and coordinated sulfation and
epimerization reactions to produce sulfated proteoglycans. Sulfated
and nonsulfated proteoglycans then travel through the Golgi network
and are ultimately secreted into the ECM by exocytosis with the
help of secretory vesicles.
Proteoglycans are heterogenous molecules, with core proteins
ranging in molecular weight from 10 kD to about 600 kD and with
attached GAG chains varying in number and type, further modified by
a complex variable pattern of sulfate groups. At least one of the
proteoglycan sugar side chains is a GAG; the core protein is
usually a glycoprotein, but may comprise up to 95% carbohydrate by
weight, mostly as long unbranched GAG chains up to at least 80
sugar residues long.
Proteoglycans along with their attached GAG chains regulate the
activities of secreted macromolecules. They can serve as selective
molecular sieves regulating a size-based trafficking of molecules
and cells, and play a role in cell-cell signaling. Proteoglycans
modulate the activities of secreted factors, such as growth factors
and cytokines, by binding to them For example, binding of
fibroblast growth factor (FGF) to heparan sulfate chains of
proteoglycans is required for FGF activation of its cell surface
receptors. On the other hand, for example, binding of a ubiquitous
growth regulatory factor, such as transforming growth factor .beta.
(TGF-.beta.) to core proteins of several ECM proteoglycans, such as
decorin, results in inhibition of TGF-.beta. activity.
Proteoglycans also bind and regulate the activities of other types
of secreted proteins, such as proteases and protease inhibitors.
Cell-surface proteoglycans also may act as co-receptors: for
example, syndecan binds to FGF and presents it to the FGF-receptor.
Similarly, betaglycan binds to TGF-.beta. and presents it to
TGF-.beta. receptors.
Collagens and elastin are the major fibrous proteins of the ECM.
Collagens comprise a family of highly characteristic fibrous
proteins and are a major component of skin and bone. Collagen
fibers consist of globular units of the collagen subunit
tropocollagen. Each tropocollagen subunit molecule comprises three
polypeptide chains, called a chains, each exhibiting a left-handed
helical conformation that are wrapped around each other in a
right-handed coiled coil structure, also called a triple helix or
super helix. A characteristic feature of collagen is a repeating
tripeptide unit comprising Glycine-Proline-X or
Glycine-X-Hydroxyproline, where X may be any amino acid. The
presence of glycine at every third position in a collagen unit is
critical for maintaining the coiled coil structure, since each
repeating glycine residue sits on the interior axis of the helix,
which sterically hinders bulkier side chains. Prolines and
hydroxyprolines help stabilize the triple helix. Collagen is
secreted as procollagen molecules, which undergo proteolytic
processing and subsequent assembly to form collagenous fibrils.
Collagens are highly glycosylated during protein trafficking
through intracellular secretory pathways.
Collagens are classified into various types depending on the nature
of their a chains. Table 1 lists types of collagen, composition,
class and distribution. (Reproduced from Shoulders and Raines,
Annu. Rev. Biochem. 2009, 78: 929-958 and Bailey's Textbook of
Microscopic Anatomy, Kelly et al., Williams and Wilkins, 18.sup.th
edition, 1984).
TABLE-US-00001 TABLE 1 Collagen Type, Class and Distribution
Collagen Type Composition Class Distribution I
.alpha..sub.1[I].sub.2.alpha..sub.2[I] Fibrillar Dermis, tendon,
ligament, bone, cornea II .alpha..sub.1[II].sub.3 Fibrillar
Cartilage, intervertebral disc, vitreous body III
.alpha..sub.1[III].sub.3 Fibrillar Fetal skin, cardiovascular
system, basal lamina, intestine. IV
.alpha..sub.1[IV].sub.2.alpha..sub.2[IV]; Network Basal lamina,
external lamina
.alpha..sub.3[IV].alpha..sub.4[IV].alpha..sub.5[IV];
.alpha..sub.5[IV].sub.2.alpha..sub.6[IV] V .alpha..sub.1[V].sub.3;
Fibrillar Bone, dermis, cornea, placenta
.alpha..sub.1[V].sub.2.alpha..sub.2[V];
A.sub.1[V].alpha..sub.2[V].alpha..sub.3[V] VI
.alpha..sub.1[VI].alpha..sub.2[VI].alpha..sub.3[VI]; Network Bone,
cartilage, cornea, dermis
.alpha..sub.1[VI].alpha..sub.2[VI].alpha..sub.4[VI] VII
.alpha..sub.1[VII].sub.2.alpha..sub.2[VII] Anchoring fibril Dermis,
bladder VIII .alpha..sub.1[VIII].sub.3; Network Dermis, brain,
heart, kidney .alpha..sub.2[VIII].sub.3;
.alpha..sub.1[VIII].sub.2.alpha..sub.2[VIII] IX
.alpha..sub.1[IX].alpha..sub.2[IX].alpha..sub.3[IX] FACIT.sup.a
Cartila- ge, cornea, vitreous X .alpha..sub.1[X].sub.3 Network
Cartilage XI .alpha..sub.1[XI].alpha..sub.2[XI].alpha..sub.3[XI]
Fibrillar Cartilage- , intervertebral disc XII
.alpha..sub.1[XII].sub.3 FACIT Dermis, tendon XIII
.alpha..sub.1[XIII].sub.3 MACIT.sup.a Endothelial cells, dermis,
eye, heart XIV .alpha..sub.1[XIV].sub.3 FACIT Bone, dermis,
cartilage XV MULTIPLEXIN.sup.a Capillaries, testis, kidney, heart,
bone XVI FACIT Dermis, kidney XVII .alpha..sub.1[XVII].sub.3 MACIT
Hemidesmosomes in epithelia XVIII MULTIPLEXIN Basal lamina, liver
XIX FACIT Basal lamina XX FACIT Cornea XXI FACIT Stomach, kidney
XXII FACIT Tissue junctions XXIII MACIT Heart, retina XXIV
Fibrillar Bone, cornea XXV MACIT Brain, heart, testis XXVI FACIT
Testis, ovary XXVII Dermis, sciatic nerve XXIX Dermis
.sup.aAbbreviations: FACIT, fibril-associated collagen with
interrupted triple helices; MACIT, membrane-associated collagen
with interrupted triple helices; MULTIPLEXIN, multiple triple helix
domains.
A network of elastic fibers in the ECM offers resilience and
elasticity so that organs are able to recoil following transient
stretch. Elastic fibers primarily comprise the fibrous protein
elastin, a highly hydrophobic protein about 750 amino acids in
length that is rich in proline and glycine, is not glycosylated and
is low in hydroxyproline and hyroxylysine. Elastin molecules are
secreted into the ECM and assemble into elastic fibers close to the
plasma membrane. Upon secretion, elastin molecules become highly
cross-linked to form an extensive network of fibers and sheets.
The ECM also comprises many non-collagen adhesive proteins, usually
with multiple domains containing binding sites of other
macromolecules and for cell-surface receptors. One such adhesive
protein, fibronectin, is a large glycoprotein comprising two
subunits joined by a pair of disulfide bonds near the carboxy
termini. Each subunit is folded into a series of rod-like domains
interspersed by regions of flexible polypeptide chains. Each domain
further comprises repeating modules of various types. One major
type of fibronectin repeating module, called type III fibronectin
repeat, is about 90 amino acids in length and occurs at least 15
times in each subunit. Fibronectin type III repeats have
characteristic Arg-Gly-Asp (RGD) tripeptide repeats that function
as binding sites for other proteins such as collagen, heparin or
cell surface receptors. Fibronectin not only plays an important
role in cell adhesion to the ECM, but also in guiding cell
migration in vertebrate embryos.
Laminin, another adhesive glycoprotein of the ECM, is a major
constituent (along with type IV collagen and another glycoprotein,
nidogen/entactin) of the basal lamina, a tough sheet of ECM formed
at the base of epithelial cells. Laminin is a large flexible
complex, about 850 kD in molecular weight, with three very long
polypeptide chains arranged in the form of an asymmetric cross held
together with disulfide bonds. Laminin contains numerous functional
domains, e.g., one binds to type IV collagen, one to heparan
sulfate, one to entactin, and two or more to laminin receptor
proteins on the cell surface.
1.2 Stem Cells
The term "stem cells" as used herein refers to undifferentiated
cells having high proliferative potential with the ability to
self-renew that can generate daughter cells that can undergo
terminal differentiation into more than one distinct cell
phenotype. Stem cells are distinguished from other cell types by
two characteristics. First, they are unspecialized cells capable of
renewing themselves through cell division, sometimes after long
periods of inactivity. Second, under certain physiologic or
experimental conditions, they can be induced to become tissue- or
organ-specific cells with special functions. In some organs, such
as the gut and bone marrow, stem cells regularly divide to repair
and replace worn out or damaged tissues. In other organs, however,
such as the pancreas and the heart, stem cells only divide under
special conditions.
Embryonic stem cells (EmSC) are stem cells derived from an embryo
that are pluripotent (i.e., they are able to differentiate in vitro
into endodermal, mesodermal and ectodermal cell types).
Adult (somatic) stem cells are undifferentiated cells found among
differentiated cells in a tissue or organ. Their primary role in
vivo is to maintain and repair the tissue in which they are found.
Adult stem cells have been identified in many organs and tissues,
including brain, bone marrow, peripheral blood, blood vessels,
skeletal muscles, skin, teeth, gastrointestinal tract, liver,
ovarian epithelium, and testis. Adult stem cells are thought to
reside in a specific area of each tissue, known as a stem cell
niche, where they may remain quiescent (non-dividing) for long
periods of time until they are activated by a normal need for more
cells to maintain tissue, or by disease or tissue injury. Examples
of adult stem cells include, but not limited to, hematopoietic stem
cells, mesenchymal stem cells, neural stem cells, epithelial stem
cells, and skin stem cells.
Hematopoietic Stem Cells (HSCs)
Hematopoietic stem cells (also known as the colony-forming unit of
the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rare
pluripotential cells within the blood-forming organs that are
responsible for the continued production of blood cells during
life. While there is no single cell surface marker exclusively
expressed by hematopoietic stem cells, it generally has been
accepted that human HSCs have the following antigenic profile: CD
34+, CD59+, Thy1+(CD90), CD38low/-, C-kit-/low and, lin-. CD45 is
also a common marker of HSCs, except platelets and red blood cells.
HSCs can generate a variety of cell types, including erythrocytes,
neutrophils, basophils, eosinophils, platelets, mast cells,
monocytes, tissue macrophages, osteoclasts, and the T and B
lymphocytes. The regulation of hematopoietic stem cells is a
complex process involving self-renewal, survival and proliferation,
lineage commitment and differentiation and is coordinated by
diverse mechanisms including intrinsic cellular programming and
external stimuli, such as adhesive interactions with the
micro-environmental stroma and the actions of cytokines.
Different paracrine factors are important in causing hematopoietic
stem cells to differentiate along particular pathways. Paracrine
factors involved in blood cell and lymphocyte formation are called
cytokines. Cytokines can be made by several cell types, but they
are collected and concentrated by the extracellular matrix of the
stromal (mesenchymal) cells at the sites of hematopoiesis. For
example, granulocyte-macrophage colony-stimulating factor (GM-CSF)
and the multilineage growth factor IL-3 both bind to the heparan
sulfate glycosaminoglycan of the bone marrow stroma. The
extracellular matrix then presents these factors to the stem cells
in concentrations high enough to bind to their receptors.
Mesenchymal Stem Cells (MSCs)
Mesenchymal stem cells (MSCs) (also known as bone marrow stromal
stem cells or skeletal stem cells) are non-blood adult stem cells
found in a variety of tissues. They are characterized by their
spindle-shape morphologically; by the expression of specific
markers on their cell surface; and by their ability, under
appropriate conditions, to differentiates along a minimum of three
lineages (osteogenic, chondrogenic, and adipogenic).
No single marker that definitely delineates MSCs in vivo has been
identified due to the lack of consensus regarding the MSC
phenotype, but it generally is considered that MSCs are positive
for cell surface markers CD105, CD166, CD90, and CD44 and that MSCs
are negative for typical hematopoietic antigens, such as CD45,
CD34, and CD14. As for the differentiation potential of MSCs,
studies have reported that populations of bone marrow-derived MSCs
have the capacity to develop into terminally differentiated
mesenchymal phenotypes both in vitro and in vivo, including bone,
cartilage, tendon, muscle, adipose tissue, and
hematopoietic-supporting stroma. Studies using transgenic and
knockout mice and human musculoskeletal disorders have reported
that MSC differentiate into multiple lineages during embryonic
development and adult homeostasis.
Analyses of the in vitro differentiation of MSCs under appropriate
conditions that recapitulate the in vivo process have led to the
identification of various factors essential for stem cell
commitment. Among them, secreted molecules and their receptors
(e.g., transforming growth factor-.beta.), extracellular matrix
molecules (e.g., collagens and proteoglycans), the actin
cytoskeleton, and intracellular transcription factors (e.g.,
Cbfa1/Runx2, PPAR.gamma., Sox9, and MEF2) have been shown to play
important roles in driving the commitment of multipotent MSCs into
specific lineages, and maintaining their differentiated
phenotypes.
For example, it has been shown that osteogenesis of MSCs, both in
vitro and in vivo, involves multiple steps and the expression of
various regulatory factors. During osteogenesis, multipotent MSCs
undergo asymmetric division and generate osteoprecursors, which
then progress to form osteoprogenitors, preosteoblasts, functional
osteoblasts, and eventually osteocytes. This progression from one
differentiation stage to the next is accompanied by the activation
and subsequent inactivation of transcription factors, i.e.,
Cbfa1/Runx2, Msx2, Dlx5, Osx, and expression of bone-related marker
genes, (i.e., osteopontin, collagen type I, alkaline phosphatase,
bone sialoprotein, and osteocalcin).
Members of the Wnt family also have been shown to impact MSC
osteogenesis. Wnts are a family of secreted cysteine-rich
glycoproteins that have been implicated in the regulation of stem
cell maintenance, proliferation, and differentiation during
embryonic development. Canonical Wnt signaling increases the
stability of cytoplasmic .beta.-catenin by receptor-mediated
inactivation of GSK-3 kinase activity and promotes .beta.-catenin
translocation into the nucleus. The active .beta.-catenin/TCF/LEF
complex then regulates the transcription of genes involved in cell
proliferation. In humans, mutations in the Wnt co-receptor LRP5
lead to defective bone formation. "Gain of function" mutation
results in high bone mass, whereas "loss of function" causes an
overall loss of bone mass and strength, indicating that Wnt
signaling is positively involved in embryonic osteogenesis.
Canonical Wnt signaling pathway also functions as a stem cell
mitogen via stabilization of intracellular .beta.-catenin and
activation of the .beta.-catenin/TCF/LEF transcription complex,
resulting in activated expression of cell cycle regulatory genes,
such as Myc, cyclin D1, and Msx1. When MSCs are exposed to Wnt3a, a
prototypic canonical Wnt signal, under standard growth medium
conditions, they show markedly increased cell proliferation and a
decrease in apoptosis, consistent with the mitogenic role of Wnts
in hematopoietic stem cells. However, exposure of MSCs to Wnt3a
conditioned medium or overexpression of ectopic Wnt3a during
osteogenic differentiation inhibits osteogenesis in vitro through
.beta.-catenin mediated down-regulation of TCF activity. The
expression of several osteoblast specific genes, (e.g., alkaline
phosphatase, bone sialoprotein, and osteocalcin), is dramatically
reduced, while the expression of Cbfa1/Runx2, an early
osteo-inductive transcription factor is not altered, implying that
Wnt3a-mediated canonical signaling pathway is necessary, but not
sufficient, to completely block MSC osteogenesis. On the other
hand, Wnt5a, a typical non-canonical Wnt member, has been shown to
promote osteogenesis in vitro. Since Wnt3a promotes MSC
proliferation during early osteogenesis, it is thought likely that
canonical Wnt signaling functions in the initiation of early
osteogenic commitment by increasing the number of osteoprecursors
in the stem cell compartment, while non-canonical Wnt drives the
progression of osteoprecursors to mature functional
osteoblasts.
Epithelial Stem Cells.
An epithelial membrane is a continuous multicellular sheet composed
of an epithelium adhered to underlying connective tissue.
Epithelial membranes can be cutaneous (e.g. skin), mucous (e.g.,
gastrointestinal lining), and or serous (e.g. pleural lining,
pericardial lining and peritoneal lining).
Epithelial stem cells line the gastrointestinal tract in deep
crypts and give rise to absorptive cells, goblet cells, paneth
cells, and enteroendocrine cells.
Components of the Human Gastrointestinal Tract
The gastrointestinal tract is a continuous tube that extends from
the mouth to the anus. On a gross level, the gastrointestinal tract
is composed of the following organs: the mouth, most of the
pharynx, the esophagus, the stomach, the small intestine (duodenum,
jejunum and ileum), and the large intestine. Each segment of the
gastrointestinal tract participates in the absorptive processes
essential to digestion by producing chemical substances that
facilitate digestion of orally-taken foods, liquids, and other
substances such as therapeutic agents.
Within the gastrointestinal tract, the small intestine, the site of
most digestion and absorption, is structured specifically for these
important functions. The small intestine is divided into three
segments: the duodenum, the jejunum, and the ileum. The absorptive
cells of the small intestine produce several digestive enzymes
called the "brush-border" enzymes. Together with pancreatic and
intestinal juices, these enzymes facilitate the absorption of
substances from the chyme in the small intestine. The large
intestine, the terminal portion of the gastrointestinal tract,
contributes to the completion of absorption, the production of
certain vitamins, and the formation and expulsion of feces.
At the cellular level, the epithelium is a purely cellular
avascular tissue layer that covers all free surfaces (cutaneous,
mucous, and serous) of the body including the glands and other
structures derived from it. It lines both the exterior of the body,
as skin, and the interior cavities and lumen of the body. While the
outermost layer of human skin is composed of dead stratified
squamous, keratinized epithelial cells, mucous membranes lining the
inside of the mouth, the esophagus, and parts of the rectum are
themselves lined by nonkeratinized stratified squamous epithelium.
Epithelial cell lines are present inside of the lungs, the
gastrointestinal tract, and the reproductive and urinary tracts,
and form the exocrine and endocrine glands.
Epithelial cells are involved in secretion, absorption, protection,
transcellular transport, sensation detection and selective
permeability. There are variations in the cellular structures and
functions in the epithelium throughout the gastrointestinal tract.
The epithelium in the mouth, pharynx, esophagus and anal canal is
mainly a protective, nonkeratinized, squamous epithelium. The
epithelium of the stomach is composed of (i) simple columnar cells
that participate in nutrient and fluid absorption and secretion,
(ii) mucus-producing goblet cells that participate in protective
and mechanical functions, and (iii) enteroendocrine cells that
participate in the secretion of gastrointestinal hormones.
Additionally, within the intestine, the epithelial lining provides
an important defense barrier against microbial pathogens.
The development of intestinal epithelium involves three major
phases: 1) an early phase of epithelial proliferation and
morphogenesis; 2) an intermediate period of cellular
differentiation in which the distinctive cell type's characteristic
of intestinal epithelium appear; and 3) a final phase of
biochemical and functional maturation. Intestinal crypts, located
at the base of villi, contain stem cells which supply the entire
epithelial cell surface with a variety of epithelial cell subtypes.
These specialized cells provide for an external
environment-internal environment interface, ion and fluid secretion
and reabsorption, antigen recognition, hormone secretion, and
surface protection. The exposure of epithelial cells on the
surfaces of the intestinal lumen subjects them to a wide range of
assaults, including microbial, chemical, and physical forces; thus
they also may contribute to patho-physiologic impairment in
diseases. Additionally, these cells are targets for inflammation,
infection, and malignant transformation.
Within the intestinal tract, the epithelium forms upon stem cell
differentiation.
Molecular Markers of Gastrointestinal Epithelial Stem Cells
As disclosed in U.S. Published Application No. 2009/0269769, which
is incorporated herein by reference in its entirety, there are no
universally accepted molecular markers that identify
gastrointestinal stem cells. However, several markers have been
used to identify stem cells in small and large intestinal tissues.
These include: .beta.-1-integrin, mushashi-1, CD45, and
cytokeratin.
CD45, also called the common leukocyte antigen, T220 and B220 in
mice, is a transmembrane protein with cytoplasmic protein tyrosine
phosphatase (PTP) activity. CD45 is found in hematopoietic cells
except erythrocytes and platelets. CD45 has several isoforms that
can be seen in the various stages of differentiation of normal
hematopoietic cells.
Mushashi-1 is an early developmental antigenic marker of stem cells
and glial/neuronal cell precursor cells.
.beta.-1-integrin (CD29, fibronectin receptor), is a .beta.-subunit
of a heterodimer protein member of the integrin family of proteins;
integrins are membrane receptors involved in cell adhesion and
recognition.
Cytokeratins are intermediate filament proteins found in the
intracytoplasmic cystoskeleton of the cells that comprise
epithelial tissue.
There are four main epithelial cell lineages: (i) columnar
epithelial cells, (ii) goblet cells, (iii) enteroendocrine
chromaffin cells, and (iv) Paneth cells. Several molecular markers
have been used to identify each of these lineages.
The markers used to identify columnar epithelial cells include:
intestinal alkaline phosphatase (ALP1), sucrase isomaltase (SI),
sodium/glucose cotransporter (SLGT1), dipeptidyl-peptidase 4
(DPP4), and CD26. Intestinal alkaline phosphatase (E.C. 3.1.3.1) is
a membrane-bound enzyme localized in the brush border of
enterocytes in the human intestinal epithelium. Sucrase-isomaltase
(SI, EC 3.2.1.48) is an enterocyte-specific small intestine
brush-border membrane disaccharidase. Dipeptidyl-peptidase 4 (E.C.
3.4.14.5) is a membrane bound serine-type peptidase. Sodium/glucose
transporter (SGLT) mediates transport of glucose into epithelial
cells. SGLT belongs to the sodium/glucose cotransporter family
SLCA5. Two different SGLT isoforms, SGLT1 and SGLT2, mediate renal
tubular glucose reabsorption in humans. Both of them are
characterized by their different substrate affinity. SGLT1
transports glucose as well as galactose, and is expressed both in
the kidney and in the intestine. SGLT2 transports glucose and is
believed to be responsible for 98% of glucose reabsorption; SGLT2
is generally found in the S1 and S2 segments of the proximal tubule
of the nephron. CD26 is a multifunctional protein of 110 KDa
strongly expressed on epithelial cells (kidney proximal tubules,
intestine, and bile duct) and on several types of endothelial cells
and fibroblasts and on leukocyte subsets.
The markers used to identify goblet cells include mucin 2 (MUC2)
and trefoil factor 3 (TFF3). Mucin-2, a secreted gel-forming mucin,
is the major gel-forming mucin secreted by goblet cells of the
small and large intestines and is the main structural component of
the mucus gel. Intestinal trefoil factor 3 is a nonmucin protein
and a product of fully differentiated goblet cells.
The markers used to identify enteroendocrine chromaffin cells
include chromogranin A (CHGA) and synaptophysin (SYP). Chromogranin
A (CHGA) and its derived peptides, which are stored and released
from dense-core secretory granules of neuroendocrine cells, have
been implicated as playing multiple roles in the endocrine,
cardiovascular, and nervous systems. Synaptophysin I (SYP) is a
synaptic vesicle membrane protein that is ubiquitously expressed
throughout the brain without a definite synaptic function.
The markers used to identify Paneth cells include lysozyme (LYZ),
defensin (DEFA1), and matrix metallopeptidase 7 (MMP7). Lysozyme
(LYZ or muramidase) (E.C. 3.2.1.17) catalyzes the hydrolysis of
1,4-beta-linkages between N-acetylmuramic acid and
N-acetyl-D-glucosamine residues in a peptidoglycan and between
N-acetyl-D-glucosamine residues in chitodextrins. Defensins (DEFA1)
are small peptides that are produced by leukocytes and epithelial
cells. Human defensin .alpha.-1 is a 3.5-kDa, 30-amino-acid peptide
that has shown effector functions in host innate immunity against
some microorganisms. Matrix metalloproteinases (MMPs) are a family
of metal-dependant enzymes that are responsible for the degradation
of extracellular matrix components. MMPs are involved in various
physiologic processes, such as embryogenesis and tissue remodeling
and also play a role in invasion and metastasis of tumor cells,
which require proteolysis of basal membranes and extracellular
matrix.
Neural Stem Cells
The adult mammalian brain contains multipotent neural stem cells
(NSCs) that have the capacity to self-renew and are responsible for
neurogenesis and maintenance of specific regions of the adult
brain. Neural stem cells can generate astrocytes, oligodendrocytes,
and neurons. Self-renewal and differentiation of neural stem cells
are directed by interactions within a complex network of intrinsic
regulators and extrinsic factors. Recent proteomic analyses have
identified a horde of transcription factors belonging to the
Wnt/.beta.-catenin, Notch and Sonic Hedgehog (shh) pathways, in
addition to epigenetic modifications, microRNA networks and
extrinsic growth factor networks, including but not limited to the
FGFs and BMPs. (Yun et al., 2010, J. Cell. Physiol. 225:
337-347).
With the advent of high throughput microarray and proteomic
technologies, a number of different molecular signatures of neural
stem cells have been identified, including but not limited to
CD133/promini, nestin, NCAM, the HMG-box transcription factor, Sox2
and the bHLH protein, Olig2. (Holmberg et al., 2011, PLoS One,
6(3): e18454; Hombach-Klonisch et al., 2008, J. Mol. Med. 86(12):
1301-1314).
Skin Stem Cells.
Several different adult stem cell populations with distinct
molecular signatures are responsible for maintaining skin
homeostasis. These include, but are not limited to, epidermal stem
cells of the interfollicular region, epidermal stem cells of the
hair follicle (also known as the bulge stem cells), dermal stem
cells, dermal papilla stem cells, and sebaceous gland stems. The
epidermal stem cells are ectodermal in origin while the dermal stem
cells originate from the mesoderm and are mesenchymal in nature.
(Zouboulis et al., 2008, Exp. Gerontol., 43: 986-997).
The interfollicular epidermal stem cells reside in the basal layer
of the epidermis and give rise to keratinocytes, which migrate to
the surface of the skin and form a protective layer. A diverse
range of molecular signatures has been described for such epidermal
stem cells including but not limited to high .alpha.6-integrin, low
CD71, high Delta 1 (Notch signaling ligand) and high CD200
expression levels. The follicular stem cells located at the base of
hair follicles give rise to both hair follicle and to the
epidermis. These are characterized by Cytokeratin 15 (K15)
immunostaining and high levels of .beta.1-integrin. Dermal stem
cell marker proteins include but are not limited to nestin,
fibronectin and vimentin, the surface markers for dermal papilla
stem cells include mesenchymal stem cell markers such as for
example CD44, CD73 and CD90 and sebaceous stem cells express
keratin 14. (Zouboulis et al., 2008, Exp. Gerontol., 43:
986-997).
In addition, adult somatic cells can be reprogrammed to enter an
embryonic stem cell-like state by being forced to express a set of
transcription factors, for example, Oct-3/4 (or Pou5f1, the Octamer
transcription factor-3/4), the Sox family of transcription factors
(e.g., Sox-1, Sox-2, Sox-3, and Sox-15), the Klf family
transcription factors (Klf-1, Klf-2, Klf-4, and Klf-5), and the Myc
family of transcription factors (e.g., c-Myc, N-Myc, and L-Myc).
For example, human inducible Pluripotent Stem cells (iPSCs) are
cells reprogrammed to express transcription factors that express
stem cell markers and are capable of generating cells
characteristic of all three germ layers (i.e., ectoderm, mesoderm,
and endoderm).
1.3. Stem Cell Niches
Adult tissue compartments contain endogenous niches of adult stem
cells that are capable of differentiating into diverse cell
lineages of determined endodermal, mesodermal or ectodermal fate
depending on their location in the body. For example, in the
presence of an appropriate set of internal and external signals,
bone marrow-derived adult hematopoietic stem cells (HSCs) have the
potential to differentiate into blood, endothelial, hepatic and
muscle cells; brain-derived neural stem cells (NSCs) have the
potential to differentiate into neurons, astrocytes,
oligodendrocytes and blood cells; gut- and epidermis-derived adult
epithelial stem cells (EpSCs) have the potential to give rise to
cells of the epithelial crypts and epidermal layers;
adipose-derived stem cells (ASCs) have the potential to give rise
to fat, muscle, cartilage, endothelial cells, neuron-like cells and
osteoblasts; and bone-marrow-derived adult mesenchymal stem cells
(MSCs) have the potential to give rise to bone, cartilage, tendon,
adipose, muscle, marrow stroma and neural cells.
Endogenous adult stem cells are embedded within the ECM component
of a given tissue compartment, which, along with support cells,
form the cellular niche. Such cellular niches within the ECM
scaffold together with the surrounding microenvironment contribute
important biochemical and physical signals, including growth
factors and transcription factors required to initiate stem cell
differentiation into committed precursors cells and subsequent
precursor cell maturation to form adult tissue cells with
specialized phenotypic and functional characteristics.
Stem Cell Markers
Coating the surface of every cell in the body are specialized
proteins ("receptors") capable of selectively binding or adhering
to other "signaling" molecules. Normally, cells use these receptors
and the molecules that bind to them as a way of communicating with
other cells and to carry out their proper functions in the body.
These cell surface receptors are the stem cell markers. Each cell
type has a certain combination of receptors on their surface that
makes them distinguishable from other kinds of cells.
The cluster of differentiation (CD) system is a protocol used for
the identification of cell surface molecules. CD molecules can act
in numerous ways, often acting as receptors or ligands; by which a
signal cascade is initiated, altering the behavior of the cell.
Some CD proteins do not play a role in cell signaling, but have
other functions, such as cell adhesion. Generally, a proposed
surface molecule is assigned a CD number once two specific
monoclonal antibodies (mAb) are shown to bind to the molecule. If
the molecule has not been well-characterized, or has only one mAb,
the molecule usually is given the provisional indicator "w."
The CD system nomenclature commonly used to identify cell markers
thus allows cells to be defined based on what molecules are present
on their surface. These markers often are used to associate cells
with certain functions. While using one CD molecule to define
populations is uncommon, combining markers has allowed for cell
types with very specific definitions. More than 350 CD molecules
have been identified for humans.
CD molecules are utilized in cell sorting using various methods,
including flow cytometry. Cell populations usually are defined
using a "+" or a "-" symbol to indicate whether a certain cell
fraction expresses or lacks a particular CD molecule.
Table 2 identifies markers commonly used to identify stem cells and
to characterize differentiated cell types:
TABLE-US-00002 TABLE 2 Commonly-Used Stem Cell Surface Markers and
Corresponding Differentiated Cell Types Marker Name Cell Type
Significance Blood Vessel Fetal liver Endothelial Cell-surface
receptor protein that identifies kinase-1 (Flk1) endothelial cell
progenitor; marker of cell-cell contacts Smooth Smooth muscle
Identifies smooth muscle cells in the wall of blood muscle cell-
vessels specific myosin heavy chain Vascular Smooth muscle
Identifies smooth muscle cells in the wall of blood endothelial
cell vessels cadherin Bone Bone-specific Osteoblast Enzyme
expressed in osteoblast; activity indicates alkaline bone formation
phosphatase (BAP) Hydroxyapatite Osteoblast Mineralized bone matrix
that provides structural integrity; marker of bone formation
Osteocalcin Osteoblast Mineral-binding protein synthesized by
osteoblast; (OC) marker of bone formation Bone Marrow and Blood
Bone Mesenchymal Important for the differentiation of committed
morphogenetic stem and mesenchymal cell types from mesenchymal stem
protein progenitor cells and progenitor cells; BMPR identifies
early receptor mesenchymal lineages (stem and progenitor cells)
(BMPR) CD4 and CD8 White blood cell Cell-surface protein markers
specific for mature T (WBC) lymphocyte (WBC subtype) CD34
Hematopoietic Cell-surface protein on bone marrow cell, indicative
stem cell (HSC), of a HSC and endothelial progenitor; CD34 also
satellite, identifies muscle satellite, a muscle stem cell
endothelial progenitor CD34.sup.+Sca1.sup.+ Mesenchymal Identifies
MSCs, which can differentiate into Lin.sup.- profile stem cell
(MSC) adipocyte, osteocyte, chondrocyte, and myocyte CD38 Absent on
HSC Cell-surface molecule that identifies WBC lineages. Present on
Selection of CD34.sup.+/CD38.sup.- cells allows for WBC lineages
purification of HSC populations CD44 Mesenchymal A type of
cell-adhesion molecule used to identify specific types of
mesenchymal cells c-Kit HSC, MSC Cell-surface receptor on BM cell
types that identifies HSC and MSC; binding by fetal calf serum
(FCS) enhances proliferation of ES cells, HSCs, MSCs, and
hematopoietic progenitor cells Colony- HSC, MSC CFU assay detects
the ability of a single stem cell or forming unit progenitor
progenitor cell to give rise to one or more cell (CFU) lineages,
such as red blood cell (RBC) and/or white blood cell (WBC) lineages
Fibroblast Bone marrow An individual bone marrow cell that has
given rise to colony-forming fibroblast a colony of multipotent
fibroblastic cells; such unit (CFU-F) identified cells are
precursors of differentiated mesenchymal lineages Hoechst dye
Absent on HSC Fluorescent dye that binds DNA; HSC extrudes the dye
and stains lightly compared with other cell types Leukocyte WBC
Cell-surface protein on WBC progenitor common antigen (CD45)
Lineage HSC, MSC Thirteen to 14 different cell-surface proteins
that are surface Differentiated markers of mature blood cell
lineages; detection of antigen (Lin) RBC and WBC Lin-negative cells
assists in the purification of HSC lineages and hematopoietic
progenitor populations Mac-1 WBC Cell-surface protein specific for
mature granulocyte and macrophage (WBC subtypes) Muc-18 Bone marrow
Cell-surface protein (immunoglobulin superfamily) (CD146)
fibroblasts, found on bone marrow fibroblasts, which may be
endothelial important in hematopoiesis; a subpopulation of Muc- 18+
cells are mesenchymal precursors Stem cell HSC, MSC Cell-surface
protein on bone marrow (BM) cell, antigen (Sca-1) indicative of HSC
and MSC Bone Marrow and Blood cont. Stro-1 antigen Stromal
Cell-surface glycoprotein on subsets of bone marrow (mesenchymal)
stromal (mesenchymal) cells; selection of Stro-1+ precursor cells,
cells assists in isolating mesenchymal precursor hematopoietic
cells, which are multipotent cells that give rise to cells
adipocytes, osteocytes, smooth myocytes, fibroblasts, chondrocytes,
and blood cells Thy-1 HSC, MSC Cell-surface protein; negative or
low detection is suggestive of HSC Cartilage Collagen types
Chondrocyte Structural proteins produced specifically by II and IV
chondrocyte Keratin Keratinocyte Principal protein of skin;
identifies differentiated keratinocyte Sulfated Chondrocyte
Molecule found in connective tissues; synthesized by proteoglycan
chondrocyte Fat Adipocyte Adipocyte Lipid-binding protein located
specifically in adipocyte lipid-binding protein (ALBP) Fatty acid
Adipocyte Transport molecule located specifically in adipocyte
transporter (FAT) Adipocyte Adipocyte Lipid-binding protein located
specifically in adipocyte lipid-binding protein (ALBP) Perilipin A
Adipocyte Protein associated with mature adipocytes Liver Albumin
Hepatocyte Principal protein produced by the liver; indicates
functioning of maturing and fully differentiated hepatocytes B-1
integrin Hepatocyte Cell-adhesion molecule important in cell-cell
interactions; marker expressed during development of liver Nervous
System CD133 Neural stem Cell-surface protein that identifies
neural stem cells, cell, HSC which give rise to neurons and glial
cells Glial fibrillary Astrocyte Protein specifically produced by
astrocyte acidic protein (GFAP) Microtubule- Neuron
Dendrite-specific MAP; protein found specifically in associated
dendritic branching of neuron protein-2 (MAP-2) Myelin basic
Oligodendrocyte Protein produced by mature oligodendrocytes;
protein (MPB) located in the myelin sheath surrounding neuronal
structures Nestin Neural Intermediate filament structural protein
expressed in progenitor primitive neural tissue Neural tubulin
Neuron Important structural protein for neuron; identifies
differentiated neuron Neurofilament Neuron Important structural
protein for neuron; identifies (NF) differentiated neuron
Neurosphere Embryoid body Cluster of primitive neural cells in
culture of (EB), ES differentiating ES cells; indicates presence of
early neurons and glia Noggin Neuron A neuron-specific gene
expressed during the development of neurons O4 Oligodendrocyte
Cell-surface marker on immature, developing oligodendrocyte O1
Oligodendrocyte Cell-surface marker that characterizes mature
oligodendrocyte Synaptophysin Neuron Neuronal protein located in
synapses; indicates connections between neurons Tau Neuron Type of
MAP; helps maintain structure of the axon Pancreas Cytokeratin 19
Pancreatic CK19 identifies specific pancreatic epithelial cells
that (CK19) epithelium are progenitors for islet cells and ductal
cells Glucagon Pancreatic islet Expressed by alpha-islet cell of
pancreas Insulin Pancreatic islet Expressed by beta-islet cell of
pancreas Insulin- Pancreatic islet Transcription factor expressed
by beta-islet cell of promoting pancreas factor-1 (PDX-1) Nestin
Pancreatic Structural filament protein indicative of progenitor
cell progenitor lines including pancreatic Pancreatic Pancreatic
islet Expressed by gamma-islet cell of pancreas polypeptide
Somatostatin Pancreatic islet Expressed by delta-islet cell of
pancreas Pluripotent Stem Cells Alkaline Embryonic stem Elevated
expression of this enzyme is associated phosphatase (ES), embryonal
with undifferentiated pluripotent stem cell (PSC) carcinoma (EC)
Alpha- Endoderm Protein expressed during development of primitive
fetoprotein endoderm; reflects endodermal differentiation (AFP)
Pluripotent Stem Cells Bone Mesoderm Growth and differentiation
factor expressed during morphogenetic early mesoderm formation and
differentiation protein-4 Brachyury Mesoderm Transcription factor
important in the earliest phases of mesoderm formation and
differentiation; used as the earliest indicator of mesoderm
formation Cluster ES, EC Surface receptor molecule found
specifically on PSC designation 30 (CD30) Cripto (TDGF- ES, Gene
for growth factor expressed by ES cells, 1) cardiomyocyte primitive
ectoderm, and developing cardiomyocyte GATA-4 gene Endoderm
Expression increases as ES differentiates into endoderm GCTM-2 ES,
EC Antibody to a specific extracellular-matrix molecule that is
synthesized by undifferentiated PSCs Genesis ES, EC Transcription
factor uniquely expressed by ES cells either in or during the
undifferentiated state of PSCs Germ cell ES, EC Transcription
factor expressed by PSCs nuclear factor Hepatocyte Endoderm
Transcription factor expressed early in endoderm nuclear factor-
formation 4 (HNF-4) Nestin Ectoderm, Intermediate filaments within
cells; characteristic of neural and primitive neuroectoderm
formation pancreatic progenitor Neuronal cell- Ectoderm
Cell-surface molecule that promotes cell-cell adhesion interaction;
indicates primitive neuroectoderm molecule (N- formation CAM)
OCT4/POU5F1 ES, EC Transcription factor unique to PSCs; essential
for establishment and maintenance of undifferentiated PSCs Pax6
Ectoderm Transcription factor expressed as ES cell differentiates
into neuroepithelium Stage-specific ES, EC Glycoprotein
specifically expressed in early embryonic embryonic development and
by undifferentiated antigen-3 PSCs (SSEA-3) Stage-specific ES, EC
Glycoprotein specifically expressed in early embryonic embryonic
development and by undifferentiated antigen-4 PSCs (SSEA-4) Stem
cell ES, EC, HSC, Membrane protein that enhances proliferation of
ES factor (SCF or MSC and EC cells, hematopoietic stem cell (HSCs),
and c-Kit ligand) mesenchymal stem cells (MSCs); binds the receptor
c-Kit Telomerase ES, EC An enzyme uniquely associated with immortal
cell lines; useful for identifying undifferentiated PSCs TRA-1-60
ES, EC Antibody to a specific extracellular matrix molecule is
synthesized by undifferentiated PSCs TRA-1-81 ES, EC Antibody to a
specific extracellular matrix molecule normally synthesized by
undifferentiated PSCs Vimentin Ectoderm, Intermediate filaments
within cells; characteristic of neural and primitive neuroectoderm
formation pancreatic progenitor Skeletal Muscle/Cardiac/Smooth
Muscle MyoD and Myoblast, Transcription factors that direct
differentiation of Pax7 myocyte myoblasts into mature myocytes
Myogenin and Skeletal Secondary transcription factors required for
MR4 myocyte differentiation of myoblasts from muscle stem cells
Myosin heavy Cardiomyocyte A component of structural and
contractile protein chain found in cardiomyocyte Myosin light
Skeletal A component of structural and contractile protein chain
myocyte found in skeletal myocyte
Table 3 shows commonly used markers employed by skilled artisans to
identify and characterize differentiated white blood cell
types:
TABLE-US-00003 TABLE 3 List of Surface Markers on White Blood Cell
Types Type of Cell CD Markers Stem cells CD34+, CD31- All leukocyte
groups CD45+ Granulocyte CD45+, CD15+ Monocyte CD45+, CD14+ T
lymphocyte CD45+, CD3+ T helper cell CD45+, CD3+, CD4+ Cytotoxic T
cell CD45+, CD3+, CD8+ B lymphocyte CD45+, CD19+ or CD45+, CD20+
Thrombocyte CD45+, CD61+ Natural killer cell CD16+, CD56+, CD3-
Table 4 correlates the exemplary protein expression profile of
adipose derived stem cells (ASCs) with the corresponding surface
markers (Flynn et. al., 2208 Organogenesis, 4(4): 228-235; Gronthos
et. al., 2011, J. Cell. Physiol., 189: 54-63).
TABLE-US-00004 TABLE 4 Adipose-derived Stem Cell Protein Expression
and Surface Marker Profile Class Protein Marker Cell Integrin
.beta..sub.1 CD29 Adhesion Integrin .alpha..sub.4 CD49.sub.d
Integrin a.sub.a CD49.sub.e Vascular Cell Adhesion Molecule VCAM;
CD106 Intracellular Adhesion Molecule -1 ICAM; CD54 Activated
Leukocyte Cell ALCAM; CD166 Adhesion Molecule Tetraspan CD9
Endoglin CD105 Muc18 CD146 Receptors Hyaluronate receptor CD44
Transferrin receptor CD71 Insulin receptor Glucocorticoid receptor
Triiodothyronine (T3) receptor Retinoic acid receptor ECM Collagen
type I Collagen type III Collagen type IV Collagen type VI CD68
Osteopontin Osteonectin Laminin Elastin Fibronectin Heparan sulfate
proteoglycan Cytoskeletal A-smooth muscle actin Vimentin Other
HLA-ABC Major histo- compatibility complex class I antigen DAF CD55
Complement protectin CD59
CD3 (TCR complex) is a protein complex composed of four distinct
chains. In mammals, the complex contains a CD3.gamma. chain, a
CD3.delta. chain, and two CD3.epsilon. chains, which associate with
the T cell receptor (TCR) and the .zeta.-chain to generate an
activation signal in T lymphocytes. Together, the TCR, the
.zeta.-chain and CD3 molecules comprise the TCR complex. The
intracellular tails of CD3 molecules contain a conserved motif
known as the immunoreceptor tyrosine-based activation motif (ITAM),
which is essential for the signaling capacity of the TCR. Upon
phosphorylation of the ITAM, the CD3 chain can bind ZAP70 (zeta
associated protein), a kinase involved in the signaling cascade of
the T cell.
Integrins are receptors that mediate attachment between a cell and
the tissues surrounding it and are involved in cell-cell and
cell-matrix interactions. In mammals, 18.alpha. and 8 .beta.
subunits have been characterized. Both .alpha. and .beta. subunits
contain two separate tails, both of which penetrate the plasma
membrane and possess small cytoplasmic domains.
Integrin .alpha.M (ITGAM; CD11b; macrophage-1 antigen (Mac-1);
complement receptor 3 (CR3)) is a protein subunit of the
heterodimeric integrin .alpha.M.beta.2 molecule. The second chain
of .alpha.M.beta.2 is the common integrin .beta.2 subunit (CD18).
.alpha.M.beta.2 is expressed on the surface of many leukocytes
including monocytes, granulocytes, macrophages and natural killer
cells. It generally is believed that .alpha.M.beta.2 mediates
inflammation by regulating leukocyte adhesion and migration.
Further, .alpha.M.beta.2 is thought to have a role in phagocytosis,
cell-mediated cytotoxicity, chemotaxis and cellular activation, as
well as being involved in the complement system due to its capacity
to bind inactivated complement component 3b (iC3b). The ITGAM
subunit of integrin .alpha.M.beta.2 is involved directly in causing
the adhesion and spreading of cells, but cannot mediate cellular
migration without the presence of the .beta.2 (CD18) subunit.
CD14 is a cell surface protein expressed mainly by macrophages and,
to a lesser extent, neutrophil granulocytes. CD14+ cells are
monocytes that can differentiate into a host of different cells;
for example, differentiation to dendritic cells is promoted by
cytokines such as GM-CSF and IL-4. CD14 acts as a co-receptor
(along with toll-like receptor (TLR) 4 and lymphocyte antigen 96
(MD-2)) for the detection of bacterial lipopolysaccharide (LPS).
CD14 only can bind LPS in the presence of lipopolysaccharide
binding protein (LBP).
CD15 (3-fucosyl-N-acetyl-lactosamine; stage specific embryonic
antigen 1 (SSEA-1)) is a carbohydrate adhesion molecule that can be
expressed on glycoproteins, glycolipids and proteoglycans. CD15
commonly is found on neutrophils and mediates phagocytosis and
chemotaxis.
CD16 is an Fc receptor (Fc.gamma.RIIIa and Fc.gamma.RIIIb) found on
the surface of natural killer cells, neutrophil polymorphonuclear
leukocytes, monocytes and macrophages. Fc receptors bind to the Fc
portion of IgG antibodies.
CD19 is a human protein expressed on follicular dendritic cells and
B cells. This cell surface molecule assembles with the antigen
receptor of B lymphocytes in order to decrease the threshold for
antigen receptor-dependent stimulation. It generally is believed
that, upon activation, the cytoplasmic tail of CD19 becomes
phosphorylated, which allows binding by Src-family kinases and
recruitment of phosphoinositide 3 (PI-3) kinases.
CD20 is a non-glycosylated phosphoprotein expressed on the surface
of all mature B-cells. Studies suggest that CD20 plays a role in
the development and differentiation of B-cells into plasma cells.
CD20 is encoded by a member of the membrane-spanning 4A gene family
(MS4A). Members of this protein family are characterized by common
structural features and display unique expression patterns among
hematopoietic cells and nonlymphoid tissues.
CD31 (platelet/endothelial cell adhesion molecule; PECAM1) normally
is found on endothelial cells, platelets, macrophages and Kupffer
cells, granulocytes, T cells, natural killer cells, lymphocytes,
megakaryocytes, osteoclasts and neutrophils. CD31 has a key role in
tissue regeneration and in safely removing neutrophils from the
body. Upon contact, the CD31 molecules of macrophages and
neutrophils are used to communicate the health status of the
neutrophil to the macrophage.
CD34 is a monomeric cell surface glycoprotein normally found on
hematopoietic cells, endothelial progenitor cells, endothelial
cells of blood vessels, and mast cells. The CD34 protein is a
member of a family of single-pass transmembrane sialomucin proteins
and functions as a cell-cell adhesion factor. Studies suggest that
CD34 also may mediate the attachment of stem cells to bone marrow
extracellular matrix or directly to stromal cells.
CD44 (the "hyaluronan receptor"), a cell-surface glycoprotein
involved in cell-cell interactions, cell adhesion and migration, is
used to identify specific types of mesenchymal cells.
CD45 (protein tyrosine phosphatase, receptor type, C; PTPRC) cell
surface molecule is expressed specifically in hematopoietic cells.
CD45 is a protein tyrosine phosphatase (PTP) with an extracellular
domain, a single transmembrane segment, and two tandem
intracytoplasmic catalytic domains, and thus belongs to receptor
type PTP. Studies suggest it is an essential regulator of T-cell
and B-cell antigen receptor signaling that functions by direct
interaction with components of the antigen receptor complexes, or
by activating various Src family kinases required for antigen
receptor signaling. CD45 also suppresses JAK kinases, and thus
functions as a regulator of cytokine receptor signaling. The CD45
family consists of multiple members that are all products of a
single complex gene. Various known isoforms of CD45 include:
CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, and
CD45R (ABC). Different isoforms may be found on different cells.
For example, CD45RA is found on naive T cells and CD45RO is found
on memory T cells.
CD56 (neural cell adhesion molecule, NCAM) is a homophilic binding
glycoprotein expressed on the surface of neurons, glia, skeletal
muscle and natural killer cells. It generally is believed that NCAM
has a role in cell-cell adhesion, neurite outgrowth, and synaptic
plasticity. There are three known main isoforms of NCAM, each
varying only in their cytoplasmic domains: NCAM-120 kDA
(glycosylphopharidylinositol (GPI) anchored); NCAM-140 kDa (short
cytoplasmic domain); and NCAM (long cytoplasmic domain). The
different domains of NCAM have different roles, with the Ig domains
being involved in homophilic binding to NCAM, and the fibronectin
type III (FNIII) domains being involved in signaling leading to
neurite outgrowth.
CD59 refers to a glycosylphosphatidylinositol (GPI)-linked membrane
glycoprotein which protects human cells from complement-mediated
lysis.
The CD66 antigen family identifies a neutrophil-specific epitope
within the hematopoietic system that is expressed by members of the
carcinoembryonic antigen family of adhesion molecules, which belong
within the immunoglobulin gene superfamily. The extracellular
portions of all CD66 (a-f) molecules possess a N-terminal V-set
IgSF domain which, lacks the canonical inter-b-sheet disulfide of
the CD-2 family. CD66a is heavily glycosylated type 1 glycoprotein
with more than 60% of the mass contributed by N-linked glycans,
which bear sialylated Lex (sLe x, CD15s) structures. In CD66a they
are spaced further apart, VxYxxLx21IxYxxV, and resemble motifs
which bind tyrosine phosphatases such as SHIP-1 and -2. Activation
of neutrophils leads to phosphorylation of tyrosine residues in the
CD66a cytoplasmic domain. CD66a is expressed on granulocytes and
epithelial cells. Products of 4 of the 7 functional
carcinoembryonic antigen (CEA) family genes, CD66a-d, are known to
be expressed on hematopoietic cells. The expression of these
molecules on hematopoietic cells is generally restricted to the
myeloid lineage. These molecules are present at low levels on
resting mature granulocytes but expression increases rapidly
following activation with inflammatory agonists, probably as a
result of exocytosis from storage granules. CD66a is detected on
some macrophages in tissue sections and has been reported on T
cells and a subpopulation of activated NK cells.
CD66b ((CGM1); CD67, CGM6, NCA-95) is a
glycosylphosphatidylinositol (GPI)-linked protein that is a member
of the immunoglobulin superfamily and carcinoembryonic antigen
(CEA)-like subfamily. CD66b, expressed on granulocytes, generally
is believed to be involved in regulating adhesion and activation of
human eosinophils.
CD90 or Thy-1 is a 25-37 kDa heavily N-glycosylated,
glycophosphatidylinositol (GPI) anchored conserved cell surface
protein with a single V-like immunoglobulin domain, originally
discovered as a thymocyte antigen. It belongs to the immunoglobulin
gene superfamily. The complex carbohydrate side chains vary in
composition between tissues and species. Generally, CD90 is
expressed on hematopoietic stem cells and neurons. CD90 is highly
expressed in connective tissue, on various fibroblast and stromal
cell lines and is expressed on all thymocytes and peripheral T
cells in mice. In humans, CD90 is expressed only on a small number
of fetal thymocytes, 10%-40% of blood CD34+ cells in bone marrow,
and <1% of CD3+CD4+ lymphocytes in peripheral circulation. CD90
also is expressed in the human lymph node HEV endothelium but not
on other endothelia and lastly, is expressed on a limited number of
lymphoblastoid and leukemic cell lines.
CD105 (endoglin) is a homodimeric integral membrane glycoprotein
composed of disulfide-linked subunits of 90-95 kDa. In humans, it
is expressed at high levels on vascular endothelial cells and on
syncytiotrophoblast of term placenta. During human heart
development, it is expressed at high levels on endocardial cushion
tissue mesenchyme during heart septation and valve formation;
subsequently expression drops as the valves mature. It also is
expressed by a population of pre-erythroblasts, leukemic cells of
lymphoid and myeloid lineages, and bone marrow stromal fibroblasts.
Endoglin is an accessory protein of multiple kinase receptor
complexes of the TGF-.beta. superfamily. The TGF-.beta.1
superfamily of structurally related peptides includes the
TGF-.beta. isoforms, .beta.1, .beta.2, .beta.3, and .beta.5, the
activins and the bone morphogenetic proteins (BMPs).
TGF-.beta.-like factors are a multifunctional set of conserved
growth and differentiation factors that control biological
processes such as embryogenesis, organogenesis, morphogenesis of
tissues like bone and cartilage, vasculogenesis, wound repair and
angiogenesis, hematopoiesis, and immune regulation. Signaling by
ligands of the TGF-.beta. superfamily is mediated by a high
affinity, ligand-induced, heteromeric complex consisting of related
Ser/Thr kinase receptors divided into two subfamilies, type I and
type II. The type II receptor transphosphorylates and activates the
type I receptor in a Gly/Ser-rich region. The type I receptor in
turn phosphorylates and transduces signals to a novel family of
recently identified downstream targets, termed Smads. Endoglin
binds transforming growth factor (TGF) TGF-.beta.1 and -.beta.3 by
associating with the TGF-.beta. type II receptor, interacts with
activin-A, interacts with bone morphogenic protein (BMP)-7 via
activin type II receptors, ActRII and ActRIIB, and binds BMP-2 by
interacting with the ligand binding type I receptors ALK3 and
ALK6.
CD166 antigen (ALCAM), a 556 amino acid glycoprotein belonging to
the immunoglobulin gene superfamily, is encoded by the activated
leukocyte-cell adhesion molecule (ALCAM) gene in humans. It
contains a secretory signal sequence, an extracellular domain which
contains 3 Ig-like C2-type domains, 2 Ig-like V-type domains and 9
potential N-linked glycosylation sites, a hydrophobic transmembrane
spanning domain and a 32 amino acid cytoplasmic domain with no
known motifs. The N-terminal Ig domain is the binding site for both
homophilic and CD166-CD6 interactions. CD166 is anchored to the
actin cytoskeleton via the cytoplasmic domain but the receptors
involved in this interaction are unknown. The soluble CD166 is
produced by proteolytic cleavage of extracellular domains or by
alternative splicing. It is expressed on mesenchymal stem cells and
progenitor cells and on cortical thymic epithelial cells and
medullary thymic epithelial cells, neurons, activated T cells, B
cells, monocytes, fibroblasts, endothelium, epithelium, primitive
subsets of hematopoietic cells including pluripotent stem cells,
blastocysts and endometrium.
1.4. Growth Factors
Growth factors are extracellular polypeptide molecules that bind to
a cell-surface receptor triggering an intracellular signaling
pathway, leading to proliferation, differentiation, or other
cellular response. These pathways stimulate the accumulation of
proteins and other macromolecules, and they do so by both
increasing their rate of synthesis and decreasing their rate of
degradation. One intracellular signaling pathway activated by
growth factor receptors involves the enzyme PI 3-kinase, which adds
a phosphate from ATP to the 3 position of inositol phospholipids in
the plasma membrane. The activation of PI 3-kinase leads to the
activation of several protein kinases, including S6 kinase. The S6
kinase phosphorylates ribosomal protein S6, increasing the ability
of ribosomes to translate a subset of mRNAs, most of which encode
ribosomal components, as a result of which, protein synthesis
increases. When the gene encoding S6 kinase is inactivated in
Drosophila, cell numbers are normal, but cell size is abnormally
small, and the mutant flies are small. Growth factors also activate
a translation initiation factor called eIF4E, further increasing
protein synthesis and cell growth.
Growth factor stimulation also leads to increased production of the
gene regulatory protein Myc, which plays a part in signaling by
mitogens. Myc increases the transcription of a number of genes that
encode proteins involved in cell metabolism and macromolecular
synthesis. In this way, it stimulates both cell metabolism and cell
growth.
Some extracellular signal proteins, including platelet-derived
growth factor (PDGF), can act as both growth factors and mitogens,
stimulating both cell growth and cell-cycle progression. This
functional overlap is achieved in part by overlaps in the
intracellular signaling pathways that control these two processes.
The signaling protein Ras, for example, is activated by both growth
factors and mitogens. It can stimulate the PI3-kinase pathway to
promote cell growth and the MAP-kinase pathway to trigger
cell-cycle progression. Similarly, Myc stimulates both cell growth
and cell-cycle progression. Extracellular factors that act as both
growth factors and mitogens help ensure that cells maintain their
appropriate size as they proliferate.
Since many mitogens, growth factors, and survival factors are
positive regulators of cell-cycle progression, cell growth, and
cell survival, they tend to increase the size of organs and
organisms. In some tissues, however, cell and tissue size also is
influenced by inhibitory extracellular signal proteins that oppose
the positive regulators and thereby inhibit organ growth. The
best-understood inhibitory signal proteins are TGF-.beta. and its
relatives. TGF-.beta. inhibits the proliferation of several cell
types, either by blocking cell-cycle progression in G1 or by
stimulating apoptosis. TGF-.beta. binds to cell-surface receptors
and initiates an intracellular signaling pathway that leads to
changes in the activities of gene regulatory proteins called Smads.
This results in complex changes in the transcription of genes
encoding regulators of cell division and cell death.
Bone morphogenetic protein (BMP), a TGF-.beta. family member, helps
trigger the apoptosis that removes the tissue between the
developing digits in the mouse paw. Like TGF-.beta., BMP stimulates
changes in the transcription of genes that regulate cell death.
Fibroblast Growth Factor (FGF)
The fibroblast growth factor (FGF) family currently has over a
dozen structurally related members. FGF1 is also known as acidic
FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 sometimes
goes by the name keratinocyte growth factor. Over a dozen distinct
FGF genes are known in vertebrates; they can generate hundreds of
protein isoforms by varying their RNA splicing or initiation codons
in different tissues. FGFs can activate a set of receptor tyrosine
kinases called the fibroblast growth factor receptors (FGFRs).
Receptor tyrosine kinases are proteins that extend through the cell
membrane. The portion of the protein that binds the paracrine
factor is on the extracellular side, while a dormant tyrosine
kinase (i.e., a protein that can phosphorylate another protein by
splitting ATP) is on the intracellular side. When the FGF receptor
binds an FGF (and only when it binds an FGF), the dormant kinase is
activated, and phosphorylates certain proteins within the
responding cell, activating those proteins.
FGFs are associated with several developmental functions, including
angiogenesis (blood vessel formation), mesoderm formation, and axon
extension. While FGFs often can substitute for one another, their
expression patterns give them separate functions. FGF2 is
especially important in angiogenesis, whereas FGF8 is involved in
the development of the midbrain and limbs.
The expression levels of angiogenic factors, such as VEGF, IGF,
PDGF, HGF, FGF, TGFm Angiopoeitin-1, and stem cell factor (SCF)
have been found to differ amongst bone-derived-,
cartilage-derived-, and adipose-derived MSCs. (Peng et al., 2008,
Stems Cells and Development, 17: 761-774).
Insulin-Like Growth Factor (IGF-1)
IGF-1, a hormone similar in molecular structure to insulin, has
growth-promoting effects on almost every cell in the body,
especially skeletal muscle, cartilage, bone, liver, kidney, nerves,
skin, hematopoietic cell, and lungs. It plays an important role in
childhood growth and continues to have anabolic effects in adults.
IGF-1 is produced primarily by the liver as an endocrine hormone as
well as in target tissues in a paracrine/autocrine fashion.
Production is stimulated by growth hormone (GH) and can be retarded
by undernutrition, growth hormone insensitivity, lack of growth
hormone receptors, or failures of the downstream signaling
molecules, including SHP2 and STAT5B. Its primary action is
mediated by binding to its specific receptor, the Insulin-like
growth factor 1 receptor (IGF1R), present on many cell types in
many tissues. Binding to the IGF1R, a receptor tyrosine kinase,
initiates intracellular signaling; IGF-1 is one of the most potent
natural activators of the AKT signaling pathway, a stimulator of
cell growth and proliferation, and a potent inhibitor of programmed
cell death. IGF-1 is a primary mediator of the effects of growth
hormone (GH). Growth hormone is made in the pituitary gland,
released into the blood stream, and then stimulates the liver to
produce IGF-1. IGF-1 then stimulates systemic body growth. In
addition to its insulin-like effects, IGF-1 also can regulate cell
growth and development, especially in nerve cells, as well as
cellular DNA synthesis.
Transforming Growth Factor beta (TGF-.beta.)
There are over 30 structurally related members of the TGF-.beta.
superfamily, and they regulate some of the most important
interactions in development. The proteins encoded by TGF-.beta.
superfamily genes are processed such that the carboxy-terminal
region contains the mature peptide. These peptides are dimerized
into homodimers (with themselves) or heterodimers (with other
TGF-.beta. peptides) and are secreted from the cell. The TGF-.beta.
superfamily includes the TGF-.beta. family, the activin family, the
bone morphogenetic proteins (BMPs), the Vg-1 family, and other
proteins, including glial-derived neurotrophic factor (GDNF,
necessary for kidney and enteric neuron differentiation) and
Mullerian inhibitory factor, which is involved in mammalian sex
determination. TGF-.beta. family members TGF-.beta.1, 2, 3, and 5
are important in regulating the formation of the extracellular
matrix between cells and for regulating cell division (both
positively and negatively). TGF-.beta.1 increases the amount of
extracellular matrix epithelial cells make both by stimulating
collagen and fibronectin synthesis and by inhibiting matrix
degradation. TGF-.beta.s may be critical in controlling where and
when epithelia can branch to form the ducts of kidneys, lungs, and
salivary glands.
The members of the BMP family were originally discovered by their
ability to induce bone formation. Bone formation, however, is only
one of their many functions, and they have been found to regulate
cell division, apoptosis (programmed cell death), cell migration,
and differentiation. BMPs can be distinguished from other members
of the TGF-.beta. superfamily by their having seven, rather than
nine, conserved cysteines in the mature polypeptide. The BMPs
include proteins such as Nodal (responsible for left-right axis
formation) and BMP4 (important in neural tube polarity, eye
development, and cell death).
Neural Epidermal Growth-Factor-Like 1 (NELL1)
Neural epidermal growth-factor-like 1 (NEL-like 1, NELL1) is a gene
that encodes an 810-amino acid polypeptide, which trimerizes to
form a mature protein involved in the regulation of cell growth and
differentiation. The neural epidermal growth-factor-like (nel) gene
first was detected in neural tissue from an embryonic chicken cDNA
library, and its human orthologue NELL1 was discovered later in
B-cells. Studies have reported the presence of NELL in various
fetal and adult organs, including, but not limited to, the brain,
kidneys, colon, thymus, lung, and small intestine.
NELL1-General Structure
Generally, the arrangement of the functional domains of the 810
amino acid NELL1 protein bears resemblance to thrombospondin-1
("THBS1") and consists of a thrombospondin N-terminal domain
("TSPN") and several von Willebrand factor, type C ("VWC"), and
epidermal growth-factor ("EGF") domains.
Additional studies have shown that there are two transcript
variants encoding different isoforms. The nel-like 1 isoform 1
precursor transcript variant represents the longer transcript and
encodes the longer isoform 1.
The conserved domains of the nel-like 1 isoform 1 precursor
transcript reside in seven regions of the isoform 1 peptide and
include: (1) a TSPN domain/Laminin G superfamily domain; (2) a VWC
domain; (3) an EGF-like domain; (4) an EGF-like domain; (5) an
EGF-like domain; (6) an EGF-like domain and (7) a VWC domain.
The first conserved domain region comprises amino acids (amino
acids 29 to 213) that are most similar to a thrombospondin
N-terminal-like domain. Thrombospondins are a family of related,
adhesive glycoproteins, which are synthesized, secreted and
incorporated into the extracellular matrix of a variety of cells,
including alpha granules of platelets following thrombin activation
and endothelial cells. They interact with a number of blood
coagulation factors and anticoagulant factors, and are involved in
cell adhesion, platelet aggregation, cell proliferation,
angiogenesis, tumor metastasis, vascular smooth muscle growth and
tissue repair. The first conserved domain also comprises amino
acids (amino acids 82 to 206; amino acids 98 to 209) that are
similar to a Laminin G-like domain. Laminin G-like (LamG) domains
usually are Ca.sup.2+ mediated receptors that can have binding
sites for steroids, .beta.1-integrins, heparin, sulfatides,
fibulin-1, and .alpha.-dystroglycans. Proteins that contain LamG
domains serve a variety of purposes, including signal transduction
via cell-surface steroid receptors, adhesion, migration and
differentiation through mediation of cell adhesion molecules.
Much of what is known about NELL1 concerns its role in bone
development. See, e.g., U.S. Pat. No. 7,884,066, U.S. Pat. No.
7,833,968, U.S. Pat. No. 7,807,787, U.S. Pat. No. 7,776,361, U.S.
Pat. No. 7,691,607, U.S. Pat. No. 7,687,462, U.S. Pat. No.
7,544,486, and U.S. Pat. No. 7,052,856, the entire contents of
which are incorporated herein by reference. It generally is
believed that during osteogenic differentiation, NELL1 signaling
may involve an integrin-related molecule and tyrosine kinases that
are triggered by NELL1 binding to a NELL1 specific receptor and a
subsequent formation of an extracellular complex. As thus far
understood, in human NELL1 (hNELL1), the laminin G domain comprises
about 128 amino acid residues that show a high degree of similarity
to the laminin G domain of extracellular matrix ("ECM") proteins,
such as human laminin .alpha.3 chain (hLAMA3), mouse laminin
.alpha.3 chain (mLAMA3), human collagen 11 .alpha.3 chain (hCOLA1),
and human thrombospondin-1 (hTSP1). This complex facilitates either
activation of Tyr-kinases, inactivation of Tyr phosphatases, or
intracellular recruitment of Tyr-phosphorylated proteins. The
ligand bound integrin (cell surface receptors that interact with
ECM proteins such as, for example, laminin 5, fibronectin,
vitronectin, TSP1/2) transduces the signals through activation of
the focal adhesion kinase (FAK) followed by indirect activation of
the Ras-MAPK cascade, and then leads to osteogenic differentiation
through Runx2; the laminin G domain is believed to play a role in
the interaction between integrins and a 67 kDa laminin
receptor.
The second conserved domain (amino acids 273 to 331) and seventh
conserved domain (amino acids 701 to 749; amino acids 703 to 749)
are similar to von Willebrand factor type C (VWC) domains, also
known as chordin-like repeats. VWC domains occur in numerous
proteins of diverse functions. It is thought that these domains may
be involved in protein oligomerization.
The third conserved domain (amino acids 434 to 471; amino acids 434
to 466), fourth conserved domain (amino acids 478 to 512), fifth
conserved domain (amino acids 549 to 586; amino acids 549 to 582),
and sixth conserved domain (amino acids 596 to 627; amino acids 596
to 634) are similar to a calcium-binding EGF-like domain.
Calcium-binding EGF-like domains are present in a large number of
membrane-bound and extracellular (mostly animal) proteins. Many of
these proteins require calcium for their biological function.
Calcium-binding sites have been found to be located at the N-term
inus of particular EGF-like domains, suggesting that
calcium-binding may be crucial for numerous protein-protein
interactions. Six conserved core cysteines form three disulfide
bridges as in non-calcium-binding EGF domains whose structures are
very similar.
The nel-like 1 isoform 2 precursor transcript variant lacks an
alternate in-frame exon compared to variant 1. The resulting
isoform 2, which has the same N- and C-term ini as isoform 1 but is
shorter compared to isoform 1, has six conserved regions including
a TSPN domain/LamG superfamily domain (amino acids 29 to 313); VWC
domains (amino acids 273 to 331; amino acids 654 to 702); and
calcium-binding EGF-like domains (amino acids 478 to 512; amino
acids 434 to 471; amino acids 549 to 580).
NELL1 and its orthologs are found across several species including
Homo sapiens (man), Mus musculus (mouse), Rattus norvegicus (rat),
Pan troglodytes (chimpanzee), Xenopus (Silurana) tropicalis (frog),
Canis lupus familiaris (dog), Culex quinquefasciatus (mosquito)
Pediculus humanus corporis (head louse), Aedes aegypti (mosquito),
Ixodes scapularis (tick), Strongylocentrotus purpuratus (purple sea
urchin), and Acyrthosiphon pisum (pea aphid).
NELL1 is Variable
NELL1 comprises several regions susceptible to increased
recombination.
Studies have indicated that susceptibilities to certain diseases
may be associated with genetic variations within these regions,
suggesting the existence of more than one causal variant in the
NELL1 gene. For example, in patients suffering irritable bowel
syndrome ("IBS"), six different single nucleotide polymorphisms
(SNPs) within NELL1 have been identified, with most of these SNPs
near the 5' end of the gene and fewer at the 3' end. These include
R136S and A153T (which reside in the TSPN) and R354W (which resides
in a VWC domain). Additional studies have identified at least 26
variants comprising some of at least 263 SNPs within the NELL1
region.
NELL1-Function
The NELL1 protein is a secreted cytoplasmic heterotrimeric protein.
The complete role NELL1 plays in vivo remains unknown.
Several studies have indicated that NELL1 may play a role in bone
formation, inflammatory bowel disease, and esophageal
adenocarcinoma, among others.
NELL1 in Osteogenesis
It generally is believed that NELL1 induces osteogenic
differentiation and bone formation of osteoblastic cells during
development. Studies have shown that the NELL1 protein (1)
transiently activates the mitogen-activated protein kinase ("MAPK")
signaling cascade (which is involved in various cellular activities
such as gene expression, mitosis, differentiation, proliferation
and apotosis); and (2) induces phosphorylation of Runx2 (a
transcription factor associated with osteoblast differentiation).
Consequently, it generally is believed that upon binding to a
specific receptor, NELL1 transduces an osteogenic signal through
activation of certain Tyr-kinases associated with the Ras-MAPK
cascade, which ultimately leads to osteogenic differentiation.
Studies have shown that bone development is severely disturbed in
transgenic mice where overexpression of NELL1 has been shown to
lead to craniosynotosis (premature ossification of the skull and
closure of the sutures) and NELL1 deficiency manifests in skeletal
defects due to reduced chondrogenesis and osteogenesis.
Additional studies have supported a role for NELL-1 as a
craniosynostosis-related gene. For example, three regions within
the NELL-1 promoter have been identified that are directly bound
and transactivated by Runx2. Further, studies in rat skullcaps have
indicated that forced expression of Runx2 induces NELL-1 expression
(which is suggestive that Nell-1 is a downstream target of
Runx2).
2. Cells of the Connective Tissue Compartment
The connective tissue compartment contains cells that primarily
function to elaborate and maintain ECM structure. The character of
the extracellular matrix is region-specific and is determined by
the amount of the extracellular materials.
Common cell types of connective tissue compartments include:
fibroblasts, macrophages, mast cells, and plasma cells. Specialized
connective tissue compartments, such as cartilage, bone, and the
vasculature, and those with special properties, such as adipose,
tendons, ligaments, etc., have specialized cells to perform
specialized functions.
2.1. Adipose Tissue Compartment
Adipose tissue compartments are dynamic, multifunctional,
ubiquitous and loose connective tissue compartments. Adipose
comprises fibroblasts, smooth muscle cells, endothelial cells,
leukocytes, macrophages, and closely packed mature lipid-filled fat
cells, termed adipocytes, with characteristic nuclei pushed to one
side, embedded within an areolar matrix that are located in
subcutaneous layers of skin and muscle (panniculus adiposus), in
the kidney region, cornea, breasts, mesenteries, mediastinium, and
in the cervical, axillary and inguinal regions. Adipocytes play a
primary role in energy storage and in providing insulation and
protection. As sites of energy storage, adipocytes regulate the
accumulation or mobilization of triacylglycerol in response to the
body's energy requirements and store energy in the form of a single
fat droplet of triglycerides, included among the more general class
of lipids.
Adipocyte Matrix
Each adipocyte is surrounded by a thick ECM called the basal
lamina. The strong adipocyte ECM scaffold lowers mechanical stress
by spreading forces over a large surface area of the adipose tissue
compartments. The ECM composition of adipocytes is similar to that
of other cell types, but it is the relative quantity of individual
components that impart cell specificity. Adipocyte ECM is
particularly enriched in collagen VI, a coiled coil comprising
.alpha.1(VI), .alpha.2(VI) and .alpha.3(VI) subunits. Collagen VI
binds to collagen IV and also to other matrix proteins such as
proteoglycans and fibronectin. Table 5 lists core proteins that
have been annotated to the adipocyte ECM with current proteomic
techniques. (Mariman et al., 2010, Cell. Mol. Life Sci.,
67:1277-1292).
TABLE-US-00005 TABLE 5 Core Proteins of Human Adipocyte ECM Protein
Symbol Basement membrane-specific heparan sulfate HSPG2
proteoglycan core protein (HSPG) (perlecan) Calreticulin CALR
Chitinase-3-like protein 1 CHI3I1 Coiled coil domain containing
protein 80 CCDC80 Collagen .alpha. 1(I) chain COL1A1 Collagen
.alpha. 2(I) chain COL1A2 Collagen .alpha. 1(III) chain COL2A1
Collagen .alpha. 2(IV) chain COL4A2 Collagen .alpha. 1(V) chain
COL5A1 Collagen .alpha. 1(VI) chain COL6A1 Collagen .alpha. 2(VI)
chain COL6A2 Collagen .alpha. 3(VI) chain COL6A3 Collagen .alpha.
1(XII) chain COL12A1 Collagen .alpha. 1(XIV) chain (undulin)
COL14A1 Collagen .alpha. 1(XV) chain COL15A1 Collagen .alpha.
1(XVIII) chain COL18A1 Decorin (bone proteoglycan II) DCN
Dermatopontin (tyrosine-rich acidic matrix protein; DPT early
quiescence protein 1) Elastin microfibril interface-located protein
1 EMILIN1 Fibronectin (FN) (cold-insoluble globulin) FN1 Fibulin-1
FBLN1 Fibulin-3 (EGF-containing fibulin-like extracellular FBLN3
matrix protein 1) Fibulin-5 (developmental arteries and neural
crest FBLN5 EGF-like protein Galectin-1 LGALS1 Galectin-3-binding
protein (lectin galactoside-binding LGALS3BP soluble 3-binding
protein) Glypican 1 GPC1 Laminin .alpha.-4 chain LAMA4 Laminin
.beta.-1 chain LAMB1 Laminin .beta.-2 chain LAMB2 Laminin .gamma.-1
chain LAMC1 Lumican (keratan sulfate proteoglycan lumican) LUM
Matrilin-2 MATN2 Microfibril-associated glycoprotein 4 MFAP4
Mimecan (osteoglycin) OGN Nidogen 1 (entactin) NID1 Nidogen 2
(osteonidogen) NID2 Periostin POSTN Proteoglycan 4 PRG4 SPARC
(osteonectin) SPARC Spondin-1 (F-spondin) (vascular smooth muscle
cell SPON1 growth-promoting factor) Spondin-2 (mindin) SPON2
Tenascin-C (TN) (hexabrachion) (cytotactin) (neuro- TNC nectin)
(GMEM) Tenascin-X TNXB Thrombospondin-1 THBS1 Thrombospondin-2
THBS2 Transforming growth factor-b-induced protein IG-H3 TGFB1
(bIG-H3) Versican core protein (large fibroblast proteoglycan)
CSPG2 Versican V3 isoform VCAN
Adipocyte ECM undergoes biphasic development during adipogenesis,
the process of formation of mature adipose tissue compartments.
There is an initial decrease in collagen I and III, whereas their
levels come back to pre-differentiation state at later stages.
Mature adipocyte ECM is maintained in a dynamic state with constant
turnover of ECM components by a balance of activities of ECM
constructive enzymes and ECM degradation enzymes. In early stages
of differentiation, the balance is shifted towards the constructive
factors. (Mariman et al., 2010, Cell. Mol. Life Sci.,
67:1277-1292). Maturation of newly synthesized ECM components is
initiated in the ER lumen where ECM proteins undergo biochemical
modifications and proteolytic processing prior to assembly. For
collagen, such modifications include proline- and
lysine-hydroxylation and glycosylation and clipping of N- and
C-terminal peptides by respective procollagen-N- and
-C-collagenase. Processed proteins are then assembled and secreted
into the extracellular environment where they undergo further
processing by secreted extracellular modification and processing
enzymes. As the preadipocytes differentiate and begin to store
fatty substances, including lipids, ECM assumes a basal laminar
structure.
Adipose-Derived Stem Cells
Adipose also comprises a population of pluripotent stem cells that
have the potential to give rise to cells of all three embryonic
lineages: ectodermal, mesodermal and endodermal. Adipogenesis,
which comprises the steps of differentiation of such pluripotent
cells to mature adipocytes, is initiated by differentiation of
these pluripotent cells to give rise to a population of mesenchymal
precursor cells or mesenchymal stem cells (MSCs), which have the
potential to differentiate into a variety of mesodermal cell
lineages such as for example, myoblasts, chondroblasts, osteoblasts
and adipocytes. In the presence of appropriate environmental and
gene expression signals, the MSCs go through growth arrest and
differentiate into precursors with a determined fate that undergo
clonal expansion, become committed and terminally differentiate to
give rise to mature cells. The population of MSCs and more
committed adipose progenitors that are found along with the stroma
of adipose tissue collectively are termed adipose-derived stem
cells (ASCs). These cells have a characteristic
CD45.sup.-CD31.sup.-CD34.sup.+CD105.sup.+ surface phenotype. In the
case of adipocyte differentiation, ASCs differentiate to
proadipocytes that undergo final differentiation to give rise to
mature adipocytes. Mesenchymal progenitor cells with chondrogenic
potential have also been identified in the infrapatellar fat pad in
joints. (Lee et al., Tissue Engg. 2010, 16(1): 317-325).
Table 6 lists cell lineages and respective inductive factors that
can be derived from ASC lines. (Brown et. al., 2010, Plast.
Reconstr. Surg., 126(6): 1936-1946; Gregoire et al., 1998, Physiol.
Rev. 78(3): 783-809).
TABLE-US-00006 TABLE 6 Inductive Factors and Cell Lineages from
Adipose-derived Stem Cells Cell Lineage Inductive Factors Adipocyte
Dexamethasone; isobutyl methylxanthine,; indoxanthine; insulin;
thiazolidinedione; nuclear hormone gluco- corticoids, e.g.,
3,3',5-triiodothyronine (T.sub.3) and retinoic acid (RA); IGF-1;
PGE.sub.2 Cardiomyocyte Transferrin; IL-3; IL-6; VEGF Chondrocyte
Ascorbic acid; bone morphogenetic protein 6; dexamethasone;
insulin; transforming growth factor-.beta. (TGF-.beta.) Endothelial
EGM-2-MV medium (Cambrex, Walkersville, MD) containing ascorbate,
epidermal growth factor, basic fibroblast growth factor, and
hydrocortisone Myocyte Dexamethasone horse serum Neuronal-like
Butylated hydroxianisole; valproic acid; insulin Osteoblast
Ascorbic acid; bone morphogenetic protein-2; dexamethasone;
1,25-dihydroxyvitamin D
Adipose Secreted Factors
Adipose is considered a secretory organ. The adipose secretome not
only includes structural and soluble factors contributing to the
formation of the adipose matrix, but also a horde of soluble
factors with endocrine function, such as growth factors, hormones,
chemokines and lipids, collectively termed adipokines. Exemplary
adipokines include, without limitation, leptin, adiponectin,
resistin, interleukin 6 (IL-6), monocyte chemoattractant protein 1
(MCP-1), tumor necrosis factor alpha (TNF-.alpha.); fibroblast
growth factor (FGF), and vascular endothelial growth factor (VEGF).
Exemplary immunogical adipokines, particularly involved in
inflammatory pathways include, without limitation, serum amyloid A3
(SAA3), IL-6, adiponectin, TNF-.alpha. and haptoglobin. Exemplary
adipokines involved in the production of new blood vessels include,
without limitation, angiopoietin-1, angiopoietin-2, VEGF,
transforming growth factor beta (TGF-.beta.), hepatic growth factor
(HGF), stromal derived growth factor 1 (SDF-1), TNF-.alpha.,
resistin, leptin, tissue factor, placental growth factor (PGF),
insulin like growth factor (IGF), and monobutyrin.
Adiponectin, a key metabolic factor secreted from adipocytes, is a
30-KDa protein that may exist as a trimer, low molecular weight
hexamers or high molecular weight 18mers. Adiponectin circulates
throughout the plasma and has a variety of metabolic effects
including, but not limited to, glucose lowering and
cardioprotection stimulation of smooth muscle proliferation.
Adiponectin has been implicated in a number of pathological
conditions including, but not limited to diabetes, obesity,
metabolic syndrome, cardiovascular disease and wound healing.
Resistin, a member of the resistin-like (RELM) hormone family, is
secreted by stromal vascular cells of adipose. Resistin is secreted
in two multimeric isoforms and functions to counterbalance the
insulin sensitizing effects of adiponectin. (Truillo, M. E. and
Scherer P. E., Endocrine Rev. 2006, 27(7): 762-778).
Secretions from resident adipocytes, macrophages and ASCs
collectively contribute to the adipose secretome. Table 7 provides
a reported adipokine profile of ASCs. (Kilroy et. al., 2007, J.
Cell. Physiol. 212: 702-709.)
TABLE-US-00007 TABLE 7 Reported Adipokine Profile of Human ASCs
Function Adipokine Angiogenic HGF VEGF Hematopoietic Flt-3 ligand
G-CSF GM-CSF IL-7 IL-12 M-CSF SCF Proinflammatory IL-1alpha IL-6
IL-8 IL-11 LIF TNF-alpha
Transcription Factors Responsible for Adipogenesis
Adipocyte differentiation involves the crosstalk between external
signals in the ECM environment with internal signals generated from
the nucleus. The peroxisome proliferator-activated receptors (PPAR)
and CCAAT-enhancer-binding proteins (C/EBP) family of transcription
factors play an important role in adipogenesis. The PPARs, members
of type II nuclear hormone receptor family, form heterodimers with
the retinoid X receptor (RXR). They regulate transcription by
binding of PPAR-RXR heteridimers to a response element
characterized by a direct repeat of the nuclear receptor hexameric
DNA recognition motif, PuGG-TCA. PPAR-.gamma. is most
adipose-specific of all PPARs and is activated prior to
transcriptional up-regulation of most other adipocyte genes. The
C/EBP family of transcription factors are also induced prior to
activation of other adipocyte genes and plays a major role in
adipocyte differentiation. Members of the basic helix-loop-helix
(bHLH) family of transcription factors have also been implicated in
adipogenesis. (Gregoire et al., 1998, Physiol. Rev. 78(3):
783-809).
2.2. Bone (Osseous) Tissue Compartment
Osseous tissue is a rigid form of connective tissue normally
organized into definite structures, the bones. These form the
skeleton, serve for the attachment and protection of the soft
parts, and, by their attachment to the muscles, act as levers that
bring about body motion. Bone is also a storage place for calcium
that can be withdrawn when needed to maintain a normal level of
calcium in the blood.
Bones can be classified according to their shape. Examples of bone
types include: long bones whose length is greater than their widths
(e.g., femur (thigh bone), humerus (long bone of the upper limb),
tibia (shin bone), fibula (calf bone), radius (the outer of the two
bones of the forearm), and ulna (inner of two bones of the
forearm)), short bones whose length and width is approximately
equal (e.g., carpals bones (wrist bones in the hand)), flat bones
(e.g., cranium (skull bones surrounding the brain), scapula
(shoulder blade), and ilia (the uppermost and largest bone of the
pelvis)), irregular bones (e.g., vertebra), and sesamoid bones,
small bones present in the joints to protect tendons (fibrous
connective tissues that connect muscles to the bones, e.g., patella
bones (knee cap)).
Grossly, two types of bone may be distinguished: cancellous,
trabecular or spongy bone, and cortical, compact, or dense
bone.
Cortical bone, also referred to as compact bone or dense bone, is
the tissue of the hard outer layer of bones, so-called due to its
minimal gaps and spaces. This tissue gives bones their smooth,
white, and solid appearance. Cortical bone consists of haversian
sites (the canals through which blood vessels and connective tissue
pass in bone) and osteons (the basic units of structure of cortical
bone comprising a haversian canal and its concentrically arranged
lamellae), so that in cortical bone, bone surrounds the blood
supply. Cortical bone has a porosity of about 5% to about 30%, and
accounts for about 80% of the total bone mass of an adult
skeleton.
Cancellous Bone (Trabecular or Spongy Bone)
Cancellous bone tissue, an open, cell-porous network also called
trabecular or spongy bone, fills the interior of bone and is
composed of a network of rod- and plate-like elements that make the
overall structure lighter and allows room for blood vessels and
marrow so that the blood supply surrounds bone. Cancellous bone
accounts for the remaining 20% of total bone mass but has nearly
ten times the surface area of cortical bone. It does not contain
haversian sites and osteons and has a porosity of about 30% to
about 90%.
The head of a bone, termed the epiphysis, has a spongy appearance
and consists of slender irregular bone trabeculae, or bars, which
anastomose to form a lattice work, the interstices of which contain
the marrow, while the thin outer shell appears dense. The irregular
marrow spaces of the epiphysis become continuous with the central
medullary cavity of the bone shaft, termed the diaphysis, whose
wall is formed by a thin plate of cortical bone.
Both cancellous and cortical bone have the same types of cells and
intercellular substance, but they differ from each other in the
arrangement of their components and in the ratio of marrow space to
bone substance. In cancellous bone, the marrow spaces are
relatively large and irregularly arranged, and the bone substance
is in the form of slender anastomosing trabeculae and pointed
spicules. In cortical bone, the spaces or channels are narrow and
the bone substance is densely packed.
With very few exceptions, the cortical and cancellous forms are
both present in every bone, but the amount and distribution of each
type vary considerably. The diaphyses of the long bones consist
mainly of cortical tissue; only the innermost layer immediately
surrounding the medullary cavity is cancellous bone. The tabular
bones of the head are composed of two plates of cortical bone
enclosing marrow space bridged by irregular bars of cancellous
bone. The epiphyses of the long bones and most of the short bones
consist of cancellous bone covered by a thin outer shell of
cortical bone.
Each bone, except at its articular end, is surrounded by a vascular
fibroelastic coat, the periosteum. The so-called endosteum, or
inner periosteum of the marrow cavity and marrow spaces, is not a
well-demarcated layer; it consists of a variable concentration of
medullary reticular connective tissue that contains osteogenic
cells that are in immediate contact with the bone tissue.
Components of Bone
Bone is composed of cells and an intercellular matrix of organic
and inorganic substances.
The organic fraction consists of collagen, glycosaminoglycans,
proteoglycans, and glycoproteins. The protein matrix of bone
largely is composed of collagen, a family of fibrous proteins that
have the ability to form insoluble and rigid fibers. The main
collagen in bone is type I collagen.
The inorganic component of bone, which is responsible for its
rigidity and may constitute up to two-thirds of its fat-free dry
weight, is composed chiefly of calcium phosphate and calcium
carbonate, in the form of calcium hydroxyapatite, with small
amounts of magnesium hydroxide, fluoride, and sulfate. The
composition varies with age and with a number of dietary factors.
The bone minerals form long fine crystals that add strength and
rigidity to the collagen fibers; the process by which it is laid
down is termed mineralization.
Bone Cells
Four cell types in bone are involved in its formation and
maintenance. These are 1) osteoprogenitor cells, 2) osteoblasts, 3)
osteocytes, and 4) osteoclasts.
Osteoprogenitor Cells
Osteoprogenitor cells arise from mesenchymal cells, and occur in
the inner portion of the periosteum and in the endosteum of mature
bone. They are found in regions of the embryonic mesenchymal
compartment where bone formation is beginning and in areas near the
surfaces of growing bones. Structurally, osteoprogenitor cells
differ from the mesenchymal cells from which they have arisen. They
are irregularly shaped and elongated cells having pale-staining
cytoplasm and pale-staining nuclei. Osteoprogenitor cells, which
multiply by mitosis, are identified chiefly by their location and
by their association with osteoblasts. Some osteoprogenitor cells
differentiate into osteocytes. While osteoblasts and osteocytes are
no longer mitotic, it has been shown that a population of
osteoprogenitor cells persists throughout life.
Osteoblasts
Osteoblasts, which are located on the surface of osteoid seams (the
narrow region on the surface of a bone of newly formed organic
matrix not yet mineralized), are derived from osteoprogenitor
cells. They are immature, mononucleate, bone-forming cells that
synthesize collagen and control mineralization. Osteoblasts can be
distinguished from osteoprogenitor cells morphologically; generally
they are larger than osteoprogenitor cells, and have a more rounded
nucleus, a more prominent nucleolus, and cytoplasm that is much
more basophilic. Osteoblasts make a protein mixture known as
osteoid, primarily composed of type I collagen, which mineralizes
to become bone. Osteoblasts also manufacture hormones, such as
prostaglandins, alkaline phosphatase, an enzyme that has a role in
the mineralization of bone, and matrix proteins.
Osteocytes
Osteocytes, star-shaped mature bone cells derived from osteoblasts
and the most abundant cell found in compact bone, maintain the
structure of bone. Osteocytes, like osteoblasts, are not capable of
mitotic division. They are actively involved in the routine
turnover of bony matrix and reside in small spaces, cavities, gaps
or depressions in the bone matrix called lacuna. Osteocytes
maintain the bone matrix, regulate calcium homeostasis, and are
thought to be part of the cellular feedback mechanism that directs
bone to form in places where it is most needed. Bone adapts to
applied forces by growing stronger in order to withstand them;
osteocytes may detect mechanical deformation and mediate
bone-formation by osteoblasts.
Osteoclasts
Osteoclasts, which are derived from a monocyte stem cell lineage
and possess phagocytic-like mechanisms similar to macrophages,
often are found in depressions in the bone referred to as Howship's
lacunae. They are large multinucleated cells specialized in bone
resorption. During resorption, osteoclasts seal off an area of bone
surface; then, when activated, they pump out hydrogen ions to
produce a very acid environment, which dissolves the hydroxyapatite
component. The number and activity of osteoclasts increase when
calcium resorption is stimulated by injection of parathyroid
hormone (PTH), while osteoclastic activity is suppressed by
injection of calcitonin, a hormone produced by thyroid
parafollicular cells.
Bone Matrix
The bone matrix accounts for about 90% of the total weight of
compact bone and is composed of microcrystalline calcium phosphate
resembling hydroxyapatite (60%) and fibrillar type I collagen
(27%). The remaining 3% consists of minor collagen types and other
proteins including osteocalcin, osteonectin, osteopontin, bone
sialoprotein, as well as proteoglycans, glycosaminoglycans, and
lipids.
Bone matrix is also a major source of biological information that
skeletal cells can receive and act upon. For example, extracellular
matrix glycoproteins and proteoglycans in bone bind a variety of
growth factors and cytokines, and serve as a repository of stored
signals that act on osteoblasts and osteoclasts. Examples of growth
factors and cytokines found in bone matrix include, but are not
limited to, Bone Morphogenic Proteins (BMPs), Epidermal Growth
Factors (EGFs), Fibroblast Growth Factors (FGFs), Platelet-Derived
Growth Factors (PDGFs), Insulin-like Growth Factor-1 (IGF-1),
Transforming Growth Factors (TGFs), Bone-Derived Growth Factors
(BDGFs), Cartilage-Derived Growth Factor (CDGF), Skeletal Growth
Factor (hSGF), Interleukin-1 (IL-1), and macrophage-derived
factors.
There is an emerging understanding that extracellular matrix
molecules themselves can serve regulatory roles, providing both
direct biological effects on cells as well as key spatial and
contextual information.
The Periosteum and Endosteum
The periosteum is a fibrous connective tissue investment of bone,
except at the bone's articular surface. Its adherence to the bone
varies by location and age. In young bone, the periosteum is
stripped off easily. In adult bone, it is more firmly adherent,
especially so at the insertion of tendons and ligaments, where more
periosteal fibers penetrate into the bone as the perforating fibers
of Sharpey (bundles of collagenous fibers that pass into the outer
circumferential lamellae of bone). The periosteum consists of two
layers, the outer of which is composed of coarse, fibrous
connective tissue containing few cells but numerous blood vessels
and nerves. The inner layer, which is less vascular but more
cellular, contains many elastic fibers. During growth, an
osteogenic layer of primitive connective tissue forms the inner
layer of the periosteum. In the adult, this is represented only by
a row of scattered, flattened cells closely applied to the bone.
The periosteum serves as a supporting bed for the blood vessels and
nerves going to the bone and for the anchorage of tendons and
ligaments. The osteogenic layer, which is considered a part of the
periosteum, is known to furnish osteoblasts for growth and repair,
and acts as an important limiting layer controlling and restricting
the extend of bone formation. Because both the periosteum and its
contained bone are regions of the connective tissue compartment,
they are not separated from each other or from other connective
tissues by basal laminar material or basement membranes. Perosteal
stem cells have been shown to be important in bone regeneration and
repair. (Zhang et al., 2005, J. Musculoskelet. Neuronal. Interact.
5(4): 360-362).
The endosteum lines the surface of cavities within a bone (marrow
cavity and central canals) and also the surface of trabeculae in
the marrow cavity. In growing bone, it consists of a delicate
striatum of myelogenous reticular connective tissue, beneath which
is a layer of osteoblasts. In the adult, the osteogenic cells
become flattened and are indistinguishable as a separate layer.
They are capable of transforming into osteogenic cells when there
is a stimulus to bone formation, as after a fracture.
Marrow
The marrow is a soft connective tissue that occupies the medullary
cavity of the long bones, the larger central canals, and all of the
spaces between the trabeculae of spongy bone. It consists of a
delicate reticular connective tissue, in the meshes of which lie
various kinds of cells. Two varieties of marrow are recognized: red
and yellow. Red marrow is the only type found in fetal and young
bones, but in the adult it is restricted to the vertebrae, sternum,
ribs, cranial bones, and epiphyses of long bones. It is the chief
site for the genesis of blood cells in the adult body. Yellow
marrow consists primarily of fat cells that gradually have replaced
the other marrow elements. Under certain conditions, the yellow
marrow of old or emaciated persons loses most of its fat and
assumes a reddish color and gelatinous consistency, known as
gelatinous marrow. With adequate stimulus, yellow marrow may resume
the character of red marrow and play an active part in the process
of blood development.
Osteogenesis or Ossification
Osteogenesis or ossification is a process by which the bones are
formed. There are three distinct lineages that generate the
skeleton. The somites generate the axial skeleton, the lateral
plate mesoderm generates the limb skeleton, and the cranial neural
crest gives rise to the branchial arch, craniofacial bones, and
cartilage. There are two major modes of bone formation, or
osteogenesis, and both involve the transformation of a preexisting
mesenchymal tissue into bone tissue. The direct conversion of
mesenchymal tissue into bone is called intramembranous
ossification. This process occurs primarily in the bones of the
skull. In other cases, mesenchymal cells differentiate into
cartilage, which is later replaced by bone. The process by which a
cartilage intermediate is formed and replaced by bone cells is
called endochondral ossification.
Intramembranous Ossification
Intramembraneous ossification is the characteristic way in which
the flat bones of the scapula, the skull and the turtle shell are
formed. In intramembraneous ossification, bones develop sheets of
fibrous connective tissue. During intramembranous ossification in
the skull, neural crest-derived mesenchymal cells proliferate and
condense into compact nodules. Some of these cells develop into
capillaries; others change their shape to become osteoblasts,
committed bone precursor cells. The osteoblasts secrete a
collagen-proteoglycan matrix that is able to bind calcium salts.
Through this binding, the prebone (osteoid) matrix becomes
calcified. In most cases, osteoblasts are separated from the region
of calcification by a layer of the osteoid matrix they secrete.
Occasionally, osteoblasts become trapped in the calcified matrix
and become osteocytes. As calcification proceeds, bony spicules
radiate out from the region where ossification began, the entire
region of calcified spicules becomes surrounded by compact
mesenchymal cells that form the periosteum, and the cells on the
inner surface of the periosteum also become osteoblasts and deposit
osteoid matrix parallel to that of the existing spicules. In this
manner, many layers of bone are formed.
Intramembraneous ossification is characterized by invasion of
capillaries into the mesenchymal zone, and the emergence and
differentiation of mesenchymal cells into mature osteoblasts, which
constitutively deposit bone matrix leading to the formation of bone
spicules, which grow and develop, eventually fusing with other
spicules to form trabeculae. As the trabeculae increase in size and
number they become interconnected forming woven bone (a
disorganized weak structure with a high proportion of osteocytes),
which eventually is replaced by more organized, stronger, lamellar
bone.
The molecular mechanism of intramembranous ossification involves
bone morphogenetic proteins (BMPs) and the activation of a
transcription factor called CBFA1. Bone morphogenetic proteins, for
example, BMP2, BMP4, and BMP7, from the head epidermis are thought
to instruct the neural crest-derived mesenchymal cells to become
bone cells directly. BMPs activate the Cbfa1 gene in mesenchymal
cells. The CBFA1 transcription factor is known to transform
mesenchymal cells into osteoblasts. Studies have shown that the
mRNA for mouse CBFA1 is largely restricted to the mesenchymal
condensations that form bone, and is limited to the osteoblast
lineage. CBFA1 is known to activate the genes for osteocalcin,
osteopontin, and other bone-specific extracellular matrix
proteins.
Endochondral Ossification (Intracartilaginous Ossification)
Endochondral ossification, which involves the in vivo formation of
cartilage tissue from aggregated mesenchymal cells, and the
subsequent replacement of cartilage tissue by bone, can be divided
into five stages. The skeletal components of the vertebral column,
the pelvis, and the limbs are first formed of cartilage and later
become bone.
First, the mesenchymal cells are committed to become cartilage
cells. This commitment is caused by paracrine factors that induce
the nearby mesodermal cells to express two transcription factors,
Pax1 and Scleraxis. These transcription factors are known to
activate cartilage-specific genes. For example, Scleraxis is
expressed in the mesenchyme from the sclerotome, in the facial
mesenchyme that forms cartilaginous precursors to bone, and in the
limb mesenchyme.
During the second phase of endochondral ossification, the committed
mesenchyme cells condense into compact nodules and differentiate
into chondrocytes (cartilage cells that produce and maintain the
cartilaginous matrix, which consists mainly of collagen and
proteoglycans). Studies have shown that N-cadherin is important in
the initiation of these condensations, and N-CAM is important for
maintaining them. In humans, the SOX9 gene, which encodes a
DNA-binding protein, is expressed in the precartilaginous
condensations.
During the third phase of endochondral ossification, the
chondrocytes proliferate rapidly to form the model for bone. As
they divide, the chondrocytes secrete a cartilage-specific
extracellular matrix.
In the fourth phase, the chondrocytes stop dividing and increase
their volume dramatically, becoming hypertrophic chondrocytes.
These large chondrocytes alter the matrix they produce (by adding
collagen X and more fibronectin) to enable it to become mineralized
by calcium carbonate.
The fifth phase involves the invasion of the cartilage model by
blood vessels. The hypertrophic chondrocytes die by apoptosis, and
this space becomes bone marrow. As the cartilage cells die, a group
of cells that have surrounded the cartilage model differentiate
into osteoblasts, which begin forming bone matrix on the partially
degraded cartilage. Eventually, all the cartilage is replaced by
bone. Thus, the cartilage tissue serves as a model for the bone
that follows.
The replacement of chondrocytes by bone cells is dependent on the
mineralization of the extracellular matrix. A number of events lead
to the hypertrophy and mineralization of the chondrocytes,
including an initial switch from aerobic to anaerobic respiration,
which alters their cell metabolism and mitochondrial energy
potential. Hypertrophic chondrocytes secrete numerous small
membrane-bound vesicles into the extracellular matrix. These
vesicles contain enzymes that are active in the generation of
calcium and phosphate ions and initiate the mineralization process
within the cartilaginous matrix. The hypertrophic chondrocytes,
their metabolism and mitochondrial membranes altered, then die by
apoptosis.
In the long bones of many mammals (including humans), endochondral
ossification spreads outward in both directions from the center of
the bone. As the ossification front nears the ends of the cartilage
model, the chondrocytes near the ossification front proliferate
prior to undergoing hypertrophy, pushing out the cartilaginous ends
of the bone. The cartilaginous areas at the ends of the long bones
are called epiphyseal growth plates. These plates contain three
regions: a region of chondrocyte proliferation, a region of mature
chondrocytes, and a region of hypertrophic chondrocytes. As the
inner cartilage hypertrophies and the ossification front extends
farther outward, the remaining cartilage in the epiphyseal growth
plate proliferates. As long as the epiphyseal growth plates are
able to produce chondrocytes, the bone continues to grow.
Bone Remodeling
Bone constantly is broken down by osteoclasts and re-formed by
osteoblasts in the adult. It has been reported that as much as 18%
of bone is recycled each year through the process of renewal, known
as bone remodeling, which maintains bone's rigidity. The balance in
this dynamic process shifts as people grow older: in youth, it
favors the formation of bone, but in old age, it favors
resorption.
As new bone material is added peripherally from the internal
surface of the periosteum, there is a hollowing out of the internal
region to form the bone marrow cavity. This destruction of bone
tissue is due to osteoclasts that enter the bone through the blood
vessels. Osteoclasts dissolve both the inorganic and the protein
portions of the bone matrix. Each osteoclast extends numerous
cellular processes into the matrix and pumps out hydrogen ions onto
the surrounding material, thereby acidifying and solubilizing it.
The blood vessels also import the blood-forming cells that will
reside in the marrow for the duration of the organism's life.
The number and activity of osteoclasts must be tightly regulated.
If there are too many active osteoclasts, too much bone will be
dissolved, and osteoporosis will result. Conversely, if not enough
osteoclasts are produced, the bones are not hollowed out for the
marrow, and osteopetrosis (known as stone bone disease, a disorder
whereby the bones harden and become denser) will result.
Bone Regeneration and Fracture Repair
A fracture, like any traumatic injury, causes hemorrhage and tissue
destruction. The first reparative changes thus are characteristic
of those occurring in any injury of soft tissue. Proliferating
fibroblasts and capillary sprouts grow into the blood clot and
injured area, thus forming granulation tissue. The area also is
invaded by polymorphonuclear leukocytes and later by macrophages
that phagocytize the tissue debris. The granulation tissue
gradually becomes denser, and in parts of it, cartilage is formed.
This newly formed connective tissue and cartilage is designated as
a callus. It serves temporarily in stabilizing and binding together
the fractured bone. As this process is taking place, the dormant
osteogenic cells of the periosteum enlarge and become active
osteoblasts. On the outside of the fractured bone, at first at some
distance from the fracture, osseous tissue is deposited. This
formation of new bone continues toward the fractured ends of the
bone and finally forms a sheath-like layer of bone over the
fibrocartilaginous callus. As the amount of bone increases,
osteogenic buds invade the fibrous and cartilaginous callus and
replace it with a bony one. The cartilage undergoes calcification
and absorption in the replacement of the fibrocartilaginous callus
and intramembraneous bone formation also takes place. The newly
formed bone is at first a spongy and not a compact type, and the
callus becomes reduced in diameter. At the time when this
subperiosteal bone formation is taking place, bone also forms in
the marrow cavity. The medullary bone growing centripetally from
each side of the fracture unites, thus aiding the bony union.
The process of repair is, in general, an orderly process, but it
varies greatly with the displacement of the fractured ends of the
bone and the degree of trauma inflicted. Uneven or protruding
surfaces gradually are removed, and the healed bone, especially, in
young individuals, assumes its original contour.
Osteogenesis and Angiogenesis
Skeletal development and fracture repair includes the coordination
of multiple events such as migration, differentiation, and
activation of multiple cell types and tissues. The development of a
microvasculature and microcirculation is important for the
homeostasis and regeneration of living bone, without which the
tissue would degenerate and die. Recent developments using in vitro
and in vivo models of osteogenesis and fracture repair have
provided a better understanding of the recruitment nature of the
vasculature in skeletal development and repair.
The vasculature transports oxygen, nutrients, soluble factors and
numerous cell types to all tissues in the body. The growth and
development of a mature vascular structure is one of the earliest
events in organogenesis. In mammalian embryonic development, the
nascent vascular networks develop by aggregation of de novo forming
angioblasts into a primitive vascular plexus (vasculogenesis). This
undergoes a complex remodeling process in which sprouting, bridging
and growth from existing vessels (angiogenesis) leads to the onset
of a functional circulatory system.
The factors and events that lead to the normal development of the
embryonic vasculature are recapitulated during situations of
neoangiogenesis in the adult. There are a number of factors
involved in neoangiogenesis; these include, but are not limited to,
Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth
Factor (bFGF), various members of the Transforming Growth factor
beta (TGF.beta.) family and Hypoxia-Inducible Transcription Factor
(HIF). Other factors that have angiogenic properties include the
Angiopoietins, (Ang-1); Hepatocyte Growth Factor (HGF);
Platelet-Derived Growth Factor (PDGF); Insulin-like Growth Factor
family (IGF-1, IGF-2) and the Neurotrophins (NGF).
The VEGFs and their corresponding receptors are key regulators in a
cascade of molecular and cellular events that ultimately lead to
the development of the vascular system, either by vasculogenesis,
angiogenesis, or in the formation of the lymphatic vascular system.
Although VEGF is a critical regulator in physiological
angiogenesis, it also plays a significant role in skeletal growth
and repair.
In the mature established vasculature, the endothelium plays an
important role in the maintenance of homeostasis of the surrounding
tissue by providing the communicative network to neighboring
tissues to respond to requirements as needed. Furthermore, the
vasculature provides growth factors, hormones, cytokines,
chemokines and metabolites, and the like, needed by the surrounding
tissue and acts as a barrier to limit the movement of molecules and
cells. Signals and attractant factors expressed on the bone
endothelium help recruit circulating cells, particularly
hematopoietic cells, to the bone marrow and coordinate with
metastatic cells to target them to skeletal regions. Thus, any
alteration in the vascular supply to bone tissue can lead to
skeletal pathologies, such as osteonecrosis (bone death caused by
reduced blood flow to bones), osteomyelitis (infection of the bone
or bone marrow by microorganism), and osteoporosis (loss of bone
density). A number of factors have been found to have a prominent
effect on the pathology of the vasculature and skeleton, including
Osteoprotegerin (OPG), which inhibits Receptor Activator of
NF-.kappa.B Ligand (RANKL)-induced osteoclastogenic bone
resorption.
Both intramembraneous and endochondral bone ossification occur in
close proximity to vascular ingrowth. In endochondral ossification,
the coupling of chondrogenesis and osteogenesis to determine the
rate of bone ossification is dependent on the level of
vascularization of the growth plate. For example, vascular
endothelial growth (VEGF) factor isoforms are essential in
coordinating metaphyseal and epiphyseal vascularization, cartilage
formation, and ossification during endochondral bone development.
HIF-1 stimulates transcription of the VEGF gene (and of other genes
whose products are needed when oxygen is in short supply). The VEGF
protein is secreted, diffuses through the tissue, and acts on
nearby endothelial cells.
The response of the endothelial cells includes at least four
components. First, the cells produce proteases to digest their way
through the basal lamina of the parent capillary or venule. Second,
the endothelial cells migrate toward the source of the signal.
Third, the cells proliferate. Fourth, the cells form tubes and
differentiate. VEGF acts on endothelial cells selectively to
stimulate this entire set of effects. Other growth factors,
including some members of the fibroblast growth factor family, also
can stimulate angiogenesis, but they influence other cell types
besides endothelial cells. As the new vessels form, bringing blood
to the tissue, the oxygen concentration rises, HIF-1 activity
declines, VEGF production is shut off, and angiogenesis ceases.
The vascularization of cartilage regions in long bones occurs at
different stages of development. In early embryonic development,
blood vessels that originate from the perichondrium invaginate into
the cartilage structures. During elevated postnatal growth,
capillaries invade the growth plate of long bones. In adulthood,
angiogenesis periodically can be switched on during bone remodeling
in response to bone trauma or pathophysiological conditions such as
rheumatoid arthritis (RA) and osteoarthritis (OA).
Bone has the unique capacity to regenerate without the development
of a fibrous scar, which is symptomatic of soft tissue healing of
wounds. This is achieved through the complex interdependent stages
of the healing process, which mimic the tightly regulated
development of the skeleton. Following trauma with damage to the
musculoskeletal system, disruption of the vasculature leads to
acute necrosis and hypoxia of the surrounding tissue. This
disruption of the circulation leads to the activation of thrombotic
factors in a coagulation cascade leading to the formation of a
hematoma. The inflammatory response and tissue breakdown activate
factors such as cytokines and growth factors that recruit
osteoprogenitor and mesenchymal cells to the fracture site. The
stimulation of the endosteal circulation in the fractured bone
allows mesenchymal cells associated with growing capillaries to
invade the wound region from the endosteum and bone marrow. At the
edge of a bone fracture, the transiently formed granulation tissue
is replaced by fibrocartilage. Concomitantly, the periosteum
directly undergoes intramembranous bone formation leading to the
formation of an external callus; while internally, the tissue is
being mineralized to form woven bone. After stabilization of the
bone tissue and vasculature in the bone fracture, the cell-mediated
remodeling cascade is activated where osteoclastic removal of
necrotic bone is followed by the replacement of the large fracture
callus by lamellar bone, the callus size is reduced, and the normal
vascular supply is restored.
A plurality of mediators associated with fetal and postnatal bone
development plays a prominent role in the cascade response in bone
fracture repair. These include but are not limited to BMP-2 and 4,
VEGF, bFGF, TGF-.beta., and PDGF. VEGF expression is detected on
chondroblasts, chondrocytes, osteoprogenitor cells and osteoblasts
in the fracture callus where it is highly expressed in angioblasts,
osteoprogenitor and osteoblast cells during the first seven days of
healing but decreases after eleven days. Additionally, osteoclasts
release heparinase that induces the release of the active form of
VEGF from heparin, activating not only angiogenesis but also
osteoclast recruitment, differentiation and activity leading to the
remodeling of the fracture callus during endochondral ossification.
Fractures in some cases fail to repair or unite resulting in
fibrous filled pseudarthrosis. A number of contributing factors can
lead to non-union or delayed union of bone fractures, such as, but
not limited to, anti-inflammatory drugs, steroids, Vitamin C,
Vitamin D and calcium deficiencies, tobacco smoking, diabetes, and
other physiological disorders.
The absence of a functional vascular network is also an important
factor in the lack of bone healing in non-union fractures. Studies
have reported that angiogenic factors released from biomimetic
scaffolds can enhance bone regeneration and that combination
strategies that release both angiogenic and osteogenic factors can
enhance the regenerative capacity of bone.
The critical sequential timing of osteoclast differentiation and
activation, angiogenesis, recruitment of osteoprogenitor cells and
the release of growth factors such as BMP-2 in osteogenesis and
fracture repair may be enhanced by the synchronized endogenous
production of angiogenic and osteogenic mediators. Studies in rat
femoral drill-hole injury have shown differential expression of
VEGF splicing isoforms along with its receptors, indicating an
important role in the bone healing process. Other studies have
demonstrated that angiogenesis occurs predominantly before the
onset of osteogenesis in bone lengthening in an osteodistraction
model.
Another angiogenic inducing growth factor, FGF-2, can accelerate
fracture repair when added exogenously to the early healing stage
of a bone. Although the mechanism has not been fully elucidated, it
has the ability to stimulate angiogenesis and the proliferation and
differentiation of osteoblasts to possibly aid the repair of bone
fractures.
2.3. Cartilaginous Tissue Compartments
Cartilaginous tissue compartments are specialized connective tissue
compartments comprising cartilage cells, known as chondrocytes,
cartilage fibers and ground substance constituting the cartilage
matrix, that collectively contribute to characteristic elastic
firmness rendering cartilage capable of withstanding high levels of
pressure or sheer. Cartilage is histologically classified into
three types depending on its molecular composition: hyaline
cartilage; fibrocartilage and elastic cartilage.
Hyaline cartilage is the predominant form of cartilage comprising
an amorphous matrix surrounding chondrocytes embedded within
spaces, known as lacunae. Hyaline cartilage, which is commonly
associated with the skeletal system and found in the nose, trachea,
bronchi and larynx, predominantly functions to provide support.
Hyaline cartilage associated with the articular portions of bone,
forming the major component of synovial joints, is termed articular
cartilage. Hyaline cartilage is usually avascular except where
vessels may pass through to supply other tissues and in
ossification centers involved in intracartilaginous bone
development.
Fibrocartilage, which is commonly found in intervertebral discs and
pubic symphysis and functions to provide tensile strength and in
shock absorption, is less firm than hyaline cartilage. It comprises
a combination of dense collagenous fibers with cartilage cells and
a scant cartilage matrix. Fibrocartilage is not usually
circumscribed by a perichondrium. Proportions of cells, fibers and
ECM components in fribrocartilage are variable.
Elastic cartilage, which is found in the external ear, the
Eustachian tube, epiglottis and some of the lanryngeal cartilages,
is characterized by a large number of elastic fibers that branch
and course in all directions to form a dense network of
anastomising and interlacing fibers.
Articular Cartilage Matrix
The chondrocytes in articular cartilage are surrounded by a narrow
region of connective tissue ECM, termed the pericellular matrix
(PCM), which together with the chondrocyte, is termed chondron. The
PCM, which is very rich in fibronectin, proteoglycans (e.g.,
aggrecan, hyaluron and decorin) and collagen (types II, VI and IX),
is particularly characterized by a high concentration of type VI
collagen as compared to the surrounding ECM. In normal articular
cartilage, type VI collagen is restricted to the chondrons, but in
osteoarthritic cartilage, it is upregulated and found throughout
the ECM. A proteomic analysis of articular cartilage revealed the
presence of collagen .alpha.1(II) C-propeptide, collagen
.alpha.1(XI) C-propeptide, collagen .alpha.2(XI) C-propeptide,
collagen .alpha.1(VI), collagen .alpha.2(VI), link protein,
biglycan, decorin, osteonectin, matrillin-1, annexin-V,
lactadherin, and binding immunoglobulin protein (BiP), in addition
to metabolic proteins. (Wilson et. al., 2008, Methods, 48:
22-31).
Chondrocyte Differentiation
The specific structure of articular cartilage, with endogenous
chondrocytes forming adult joints, is the result of endochondral
ossification, as described above under the heading, Osseous Tissue
Compartments Formation.
Chondrocyte differentiation and maintenance in articular cartilage
is governed by interaction of multiple factors. Key players
include, but are not limited to, ions (e.g., calcium); steroids
(e.g., estrogens); terpenoids (e.g., retinoic acid); peptides
(e.g., Parathyroid hormone (PTH), parathyroid hormone-related
peptide (PTHrP)), insulin growth factors (e.g., TGF.beta. hormones,
including, without limitation, BMPs, IGF-1, VEGF, PDGF, FGF);
transcription factors (e.g., Wnt, SOX-9); eicosanoids (e.g.,
prostaglandins); catabolic interleukins (e.g., IL-1); and anabolic
interleukins (e.g., IL-6, IL-4 and IL-10). (Gaissmaier et al.,
2008, Int. J. Care Injured, 39S1: S88-S96).
Growth Plate
The epiphyseal plates or growth plates are a hyaline cartilage
plate located in the metaphysis at the end of long bones. Whereas
endochondral ossification is responsible for the formation of
cartilage in utero and in infants, the growth plates are
responsible for the longitudinal growth of long bones via a
cartilage template. The ongoing developmental processes of
proliferation and differentiation within the growth plates are
mediated by a number of hormonal and paracrine factors secreted by
the growth plate chondrocytes. The growth plate is a highly
organized structure comprising a large number of chondrocytes in
various stages of differentiation and proliferation embedded in a
scaffold of ECM components.
The growth plate can be subdivided into four zones depending on the
stage of differentiation and spatial distribution of collagen
types. The resting zone is the smallest zone close to the
epiphyseal cartilage comprising small monomorphic chondrocytes with
a narrow rim of cytoplasm. The chondrocytes of the resting zone
secrete growth plate orienting factor (GPOF) that aligns
proliferating cells parallel to the long axis of the developing
bone. Stem cell-like cells of the resting zone have a limited
proliferative capacity, which eventually leads to fusion of the
growth plate (epiphyseal fusion). The proliferative zone of the
growth plate comprises chondrocytes that are arranged in
characteristic columns parallel to the longitudinal axis of the
bone and are separated by ECM with high type II collagen. The
chondrocytes of the proliferative zone are mitotically active, have
high oxygen and glycogen content, and exhibit increased
mitochondrial ATP production. The hypertrophic zone refers to the
zone farthest from the resting zone where prehypertrophic
chondrocytes stop dividing and terminally differentiate into
elongated hypertrophic chondrocytes embedded in ECM high in type X
collagen. Hypertrophic chondrocytes have a high intracellular
calcium concentration required for the production of release
vesicles containing Ca.sup.2+-binding annexins, that secrete
calcium phosphate, hydroxyapatite, phosphatases (such as alkaline
phosphatase), metalloproteinases, all instrumental in proteolytic
remodeling and mineralization of the surrounding matrix. The
hypertrophic chondrocytes produce factors, such as VEGF, that
initiate vascularization of the mineralized matrix that is then
degraded by invading phagocytic chondroclasts and osteoclasts
constituting the invading zone.
The developmental processes involving chondrogenesis are regulated
by an interplay of a large number of systemic hormones and
paracrine factors, including growth factors, cytokines and
transcription factors. Table 8 lists key factors involved in
chondrocyte proliferation and differentiation in the growth plate.
(Brochhausen et al., J. Tissue Eng. Regen. Med. 2009, 3:
416-429).
TABLE-US-00008 TABLE 8 Summary of Key Factors involved in
Chondrocyte Proliferation and Differentiation in the Growth Plate
Name Class Expression Effect ATF-2 Transcription Resting
chondrocytes; Apoptosis factor proliferative chondrocytes Bcl-2
Inner mitochondrial Proliferative Apoptosis membrane protein
chondrocytes; prehypertrophic chondrocytes Ihh Signaling molecule
Prehypertrophic Proliferation chondrocytes PTHrP Peptide hormone
Perichondrium Proliferation perarticular chondrocytes BMP
TGF-.beta. superfamily Prehypertrophic Cartilage growth factors
chondrocytes formation; proliferation PGE.sub.2 Lipid mediator All
zones of Proliferation growth plate matrix synthesis MMP
Metalloproteinase Hypertrophic Apoptosis; chondrocytes;
vascularization chondroclasts matrix degradation Sox Transcription
Resting and Differentiation; factor proliferative proliferation;
chondrocytes; hypertrophic chondrocytes Runx 2 Transcription
Hypertrophic Terminal (Cbfa 1) factor chondrocytes differentiation;
matrix mineralization NOTCH Single pass Prehypertrophic Inhibits
transmembrane and hypertrophic terminal protein chondrocytes
differentiation HOX Homeobox Hypertrophic Activates transcription
chondrocytes osteogenic factors genes FGF Fibroblast growth
Proliferative Anti- factor chondrocytes proliferation
Stem Cells of Cartilaginous Tissue Compartments
Multipotent mesenchymal progenitor cells with adipogenic,
osteogenic and chondrogenic potential, and that are
CD105+/CD166+(corresponding to TGF-.beta. type III receptor
(endoglin) and ALCAM, respectively), have been identified in
articular cartilage. (Asalameh et al., Arthritis & Rheumatism,
2004, 50(5): 1522-1532). The presence of
CD34-/CD45-/CD44+/CD73+/CD90+ mesenchymal stem cells with
adipogenic, chondrogenic and osteogenic potential also has been
shown. (Peng et al., Stem Cells and Development (2008), 17:
761-774). Similar to bone-derived MSCs, articular-derived MSCs are
positive for surface expression of Notch-1. (Hiraoka et al.,
Biorheology, 2006, 43: 447-454). A potential MSC niche positive for
Stro-1, Jagged-1 and BMPr1a has also been identified in the
perichondrial zone of Ranvier on the growth plate. (Karlsson et
al., 2009, J. Anat. 215(3): 355-63).
Differential expression of Notch-1, Stro-1 and VCAM-1/CD106 markers
has been observed in normal articular cartilage versus
osteoarthritic (OA) cartilage. In normal cartilage, expression of
these markers is higher in the superficial zone (SZ) as compared to
the middle zone (MZ) and deep zone (DZ). On the other hand, OA
cartilage SZ has reduced Notch-1 and Sox-9 while MZ has increased
Notch-1, Stro-1 and VCAM-1 positive cells. (Grogan et al.,
Arthritis Res. Ther. 2009, 11(3): R85-R97).
Intervertebral Disc Fibrocartilage Tissue Compartments
The intervertebral discs (IVD) predominantly are comprised of
fibrocartilage. The IVD fibrocartilage is continuous both with and
below the articular cartilage of adjacent vertebrae as well as
peripherally with spinal ligaments. The IVD is a unique structure
containing annulus fibrosus (AF) and nucleus pulposus (NP), a
gelatinous ellipsoidal remnant of the embryonic notochord, and is
sandwiched between two adjacent cartilaginous endplates (EP). IVD
rupture and herniation of the nucleus pulposus into the spinal cord
may cause severe pain and other neurological symptoms. The NP and
AF synergistically function to achieve the primary role of IVD in
transferring load, dissipating energy and facilitating in joint
mobility.
The adult IVD is essentially avascular; hence, endogenous cells
survive in a low-nutrient and low-oxygen microenvironment. The
major ECM components of IVD include but are not limited to
aggrecan, collagen (e.g., types I, II and IX), leucine rich repeat
(LRR) proteins and proteoglycans (e.g., fibromodulin, decorin,
lumican), cartilage oligomatrix protein, and collagen VI beaded
filament network. (Feng et al., 2006, J. Bone Joint Surg. Am. 88:
25-29). The water content, GAG content, aggrecan levels and levels
of type II collagen are significantly lower in older discs
demonstrating the effects of IVD degeneration with age. (Murakami
et al., 2010, Med. Biol. Eng. Comput. 48: 469-474).
The central nucleus pulposus (NP) is rich in aggrecan and hyaluron.
The developing NP is characterized by the presence of highly
vacuolated chondrocytes and small chondroblasts inherited from the
notochord. Primarily functioning as a primitive axial support, the
integrity of the notochord is maintained by a proteoglycan (PG-)
and laminin-rich sheath. As NP matures, the cellular composition
becomes predominantly chondrocytic. Mature NP cells are small and
have an aggrecan rich matrix, which is essential in maintaining
requisite hydration levels for mechanical function. Their gene
expression profile and metabolic activity are distinct from the
chondrocytes of articular cartilage. The ECM of immature NP has
high aggrecan levels and primarily contains type II collagen, with
the type IIA isoform expressed by progenitor cells during
chondrogenesis, not by mature chondrocytes. (Hsieh A. H. and
Tworney J. D., J. Biomech., 2010, 43(1): 137-156).
The AF surrounds the NP with layers of unidirectional sheets of
collagen parallel to the circumference of a disc to form collagen
lamellae. Alternating bidirectional collagen fibers intersperse the
AF collagen lamellae. AF can be subdivided into three regions:
inner AF, middle AF and outer AF. The inner AF arises along with
endochondral formation of the vertebrae. The outer AF arises as a
separate cell condensation with slower matrix formation. Lamellae
of inner AF comprises predominantly of type II collagen and
fibrochondrocytes, while those of outer AF are comprised of type I
collagen and fibroblasts. A population of pancake shaped
interlamellar cells as well as elastin fibers are also found within
the lamellae, in vertebral attachments, and at the NP-AF interface.
Large proteoglycans (PGs; for example aggrecan and versican) and
type I and VI collagen permeate interlamellar and translamellar
ECM. (Hsieh A. H. and Tworney J. D., J. Biomech., 2010, 43(1):
137-156).
A large number of coordinated signals originating from the cells of
the notochord and floor plate of the embryonal neural tube are
instrumental in disc embryogenesis. Key signals include, but are
not limited to, sonic hedgehog (Shh), Wnt, noggin, Pax family of
transcription factors (e.g., Pax 1 and Pax 9), Sox family of
transcription factors (Sox5, Sox6 and Sox) and TGF-.beta.. (Smith
et al., 2011, Dis Model Mech. 4(1): 31-41). Herniation and IVD
degeneration are associated with changes in inflammatory and immune
cytokine profiles, including, but not limited to, the activation of
Th1-related cytokines (e.g. IFN.gamma.) as well as Th17-related
cytokines (e.g., IL-4, IL-6, IL-12 and IL-17). (Shamji et al.,
2010, Arthritis & Rheumatism, 62(7): 1974-1982).
A potential stem cell niche comprised of progenitor cells that are
positive for Notch1, Delta4, Jagged1, CD117, Stro-1 and Ki67 has
been identified in intervertebral discs of a number of animals,
including humans. It has been reported that the IVD tissue
compartments comprise a slow growing zone in the AF as well as the
NP regions. (Henriksson et al., 2009, SPINE, 34(21):
2278-2287).
2.4. Dental Tissue Compartments
A tooth has three anatomical divisions (crown, root and neck), and
four structural components (enamel, dentin, cementum and pulp).
Enamel is the hardest, most mineralized biological tissue in the
human body. It is composed of elongated hydroxyapatite crystallites
bundled into rods or prisms, interspersed with crystalline
interrods filling the interstitial space. Enamel cells, known as
ameloblasts, are responsible for enamel development. Ameloblastin,
TRAP and enamelin are key proteins found in enamel tissue whereas
the enamel matrix is devoid of collagen, composed primarily of
amelogenin. An intricate orchestration of signaling factors, such
as BMPs (e.g., BMP-2, BMP-4, BMP-7), FGFs (e.g., FGF-3, -4, -9,
-20), Wnt-3, 10a, 10b and transcription factors, such as, p21, Msx2
and Lef1 is responsible for morphogenesis of enamel. Self-assembly
of amelogens to form amelogenin nanospheres play a role in
nucleation of hydroxyapatite crystallization and enamel
mineralization. Matrix processing enzymes, such as MMP-20,
kallikrein-4 (KLK4), also known as enamel matrix serine protease-1
(EMSP-1), are involved in the complete elimination of the protein
matrix and replacement with a mineralized matrix. (Fong et al.,
2005, J. Dent. Educ., 69(5): 555-570). Ameloblasts arise from
epithelial stem cells of ectodermal origin. They are lost after
tooth eruption leaving no adult human ectodermal stem cells in the
mature enamel. In contrast, rodent enamel retain a niche of
epithelial stem cells, known as apical bud cells, for continuous
enamel production. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed,
120:860-872).
Dentin is a hard, yellowish and elastic living connective tissue
compartment with biomechanical properties similar to bone. The
formation of dentin is driven by mesenchymally derived mature
odontoblasts that are fully differentiated and nondividing and that
form a single layer underneath the dentin in a mature tooth. A
series of epithelial-mesenchymal interactions regulates odontoblast
differentiation from neural crest cells in the first branchial arch
and frontonasal processes. Mature dentin is comprised of a mantle,
composed of intertubular and peritubular dentin made of a collagen
fibril matrix, with odontoblast cell processes extending into
dentin tubules. During dentinogenesis, odontoblasts secrete
predentin, a mineralized tissue composed of type I collagen. Unlike
osteogenesis, in dentinogenesis, as the predentin layer is formed,
the odontoblasts recede instead of becoming embedded within the
dentin matrix, leaving behind cells processes within dentinal
tubules. Subsequently, the unmineralized predentin is converted to
dentin by gradual mineralization of collagen. Dentinogenesis is
directed by a series of highly controlled biochemical events that
control the rates of collagen secretion, its maturation into thick
fibrils, loss of proteoglycans, mineral formation including hydroxy
apatite crystallization, and growth. The dentin matrix is primarily
composed of collagens (e.g., types I, III and V) as well as other
matrix proteins, including, but not limited to, phosphorylated and
nonphosphorylated matrix proteins, proteoglycans, growth factors,
metalloproteinases, alkaline phosphatase serum derived proteins,
and phospholipids. (Fong et al., 2005, J. Dent. Educ., 69(5):
555-570). No stem cells have been identified in mature dentin.
The dental pulp is the tooth's living tissue that responds to pain
and damage and initiates tissue repair. An odontoblast cell layer
forms the outer boundary of the pulp and is associated with an
underlying network of dendritic cells. A cell-free zone underlying
the odontoblast layer is rich in nerve fibers and blood vessels.
Similar to dentin, dental pulp also differentiates from neural
crest-derived ectomesenchyme during tooth development.
Several sources of stem cells have been identified associated with
pulp tissue. In immature teeth, apical papilla, the embryonal organ
responsible for pulp differentiation, is the source for stem cells
of apical papilla (SCAP). Mature dental pulp is the source of
dental pulp stem cells (DPSC) whereas stem cells are also extracted
from exfoliated deciduous teeth (SHED). Additional cells of the
dental pulp core that function in pulpal defense, include, but are
not limited to, macrophages, lymphocytes and mast cells. Pulp
matrix is composed of collagens (e.g., types I, III, V and VI), but
lacks mineralization. Other noncollagenous proteins of the pulp
matrix are similar in composition to dentin. The dental pulp is
capable of responding to dentin tissue damage by secreting new
dentin from old odontoblast populations or generation and secretion
of dentin from new secondary odontoblast populations. (Fong et al.,
2005, J. Dent. Educ., 69(5): 555-570).
The periodontium consists of tissues supporting the tooth crown,
including a nonmineralized periodontal ligament (PDL) sandwiched
between layers of mineralized tissues, including the cementum,
alveolar bone and dentin. Cementum is a thin mineralized layer
covering the dentin. Cementoblasts are cells responsible for
cementum matrix secretion and subsequent mineralization. When
cementoblasts become entrapped within cementum matrix, they are
termed cementocytes. Cementoblasts are ectomesenchymal, being
derived from neural crest cells, similar to PDL and alveolar bone.
Like bone and dentin, cementum is a collagenous mineralized tissue
that hardens upon formation of carbonated hydroxyapatite. (Fong et
al., 2005, J. Dent. Educ., 69(5): 555-570).
PDL is a space between cementum and alveolar bone. It represents a
replacement of the dental follicle region in immature developing
teeth. Mature PDL contains mostly periodontal fibroblasts as well
as stem cells, known as the periodontal ligament stem cells
(PDLSCs). The immature dental follicle is also a source of
mesenchymal stem cells, known as dental follicle stem cells
(DFSCs). (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).
Table 9 shows the differentiation potential of dental mesenchymal
cells. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed,
120:860-872).
TABLE-US-00009 TABLE 9 Differentiation Potential Dental Mesenchymal
Stem Cells DPSC SHED PDLSC DFSC SCAP Adipocytes X X X X
Cementoblasts X X Chondrocytes X X Dental pulp X Dentin X
Endothelocytes X X Musculature X Neuroblasts X Neurons X X
Odontoblasts X X X X Osteoblasts X X X X X PDL X Progenitors
Periodontium X
Several dental stem cell markers have been identified. Stro-1 and
Stro-4 are commonly used dental stem cell markers for all dental
mesenchymal stem cells. Dental stem cells originating from the
neural crest have the neural marker, nestin. An osteoblast marker,
osteocalcin, is also used as a stem cell marker for DPSCs.
Similarly, SCAPs express Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and
TRA-1-81. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed,
120:860-872).
2.5. Fascial Tissue Compartment
Fascial tissue compartments form a layer of fibrous tissue found
throughout the body surrounding softer and more delicate organs,
including but not limited to muscles, groups of muscles, blood
vessels, nerves, etc. Fascial tissue originates from the embryonic
mesenchyme. Fasciae form during the development of bones, muscles
and vessels from the mesodermal layer of the embryo. Fascial tissue
can be categorized into three types depending on location: (1)
superficial fascial tissue, which is found beneath the integument
throughout the body, usually blending with the reticular layer of
the dermis; (2) deep fascial tissue comprising dense fibroareolar
connective tissue surrounding muscles, bones, nerves and blood
vessels; and (3) visceral or subserous fascia, which suspends
organs within their cavities and wraps them in layers of connective
tissue membranes. (Chapter IV. Myology, Section 3. Tendons,
Aponeuroses, and Fasciae, Gray's Anatomy of the Human Body,
20.sup.th Edition, Re-edited by Lewis, W. H., Lea & Febiger,
Philadelphia, 1918, Bartleby.com, New York, 2000).
The fibroareolar connective tissue of fascia comprises four kinds
of cells: (1) flattened lamellar cells, which may be branched or
unbranched (branched lamellar cells contain clear cytoplasm and
oval nuclei and project multidirectional processes that may unite
to form an open network, such as in the cornea; unbranched lamellar
cells are joined end to end. (2) Clasmatocytes, which are large
irregular vacuolated or granulated cells with oval nuclei. (3)
Granule cells, which are ovoid or spherical in shape. (4) Plasma
cells of Waldeyer, usually spheroidal, characterized by vacuolated
protoplasm.
2.6. Ligament Tissue Compartment
The term "ligaments" as used herein refers to dense regular
connective tissue comprising attenuated collagenous fibers that
connect bones at joints. Ligament ECM is composed of type I and
type III collagens together with other proteoglycans and
glycoproteins. Mesenchymal stem cells have been found in the human
anterior cruciate ligament that exhibit multilineage
differentiation potential, like bone-derived mesenchymal stem
cells. (Cheng et al., 2010, Tissue Engg. A, 16(7):2237-2253).
2.7. Synovial Tissue Compartment
The synovial membrane is composed of fibrous connective tissue and
lines the joint cavity of synovial joints. It is made up of a layer
of macrophage (type A) and fibroblast-like (type B) synoviocytes
and a loose sublining tissue. Synovial fluid is secreted by
synovial cells lining the synovial membrane in the joint capsule.
It is a viscid, mucoalbuminous fluid, rich in hyaluronic acid. It
acts as a lubricating fluid, facilitating the smooth gliding of the
articular surface. Functional mesenchymal stem cell niches have
been identified as resident to synovial lining and subsynovial
tissue. These cells are positive for the artificial nucleoside,
iododeoxyuridine (IdU) as well as MSC markers such as PDGFR.alpha.,
p75 and CD44 and have chondrogenic potential. (Kurth et al.,
Arthritis Rheum., 2011, 63(5): 1289-1300). Synovial fluid-derived
MSCs have also been identified, and these have higher chondrogenic
potential as compared to bone marrow-derived and adipogenic MSCs.
(Koga et al., 2008, Cell Tissue Res., 333: 207-215). Synovial MSCs
and MPCs have been shown to prevent degeneration due to
intervertebral disc disease (IVD) and to be useful for cartilage
tissue engineering. (Miyamoto et al., 2010, Arthritis Res. Ther.,
12: R206-218; Lee et al., 2010, Tissue Engg. A, 16(1):
317-325).
2.8. Tendon Tissue Compartment
Tendons are specialized connective tissue compartments that connect
bone to muscle. Tendon cells are embedded amongst a parallel group
of collagenous fibers that secrete a unique ECM containing
collagens, large proteoglycans, and small leucine rich
proteoglycans that function as lubricators and organizers of
collagen fibril assembly. A unique tendon stem/progenitor cell
(TSPC) niche has been identified amongst the parallel collagen
fibrils surrounded by ECM. The TSPCs exhibit osteogenic and
adipogenic potential. Biglycan and fibromodulin are key tendon ECM
components that direct TSPC fate through BMP signaling. These TSPCs
are positive for bone marrow derived stem cell markers such as
Stro-1, CD146, CD90 and CD44 but not for CD18. TSPCs do not express
hematopoietic markers, such as CD34, CD45 and CD117, or the
endothelial marker CD106. (Bi et al., 2007, Nat. Med., 13(10):
1219-1227).
2.9. Vasculature Tissue Compartment
The vascular wall is made of three concentric zones with distinct
cellular composition, all mesodermal in origin: the tunica intima,
containing predominantly mature differentiated endothelial cells
(EC), the tunica media, containing mature and differentiated smooth
muscle cells, and the tunica adventitia, containing mature
fibroblasts. (Tilki et al., 2009, Trends Mol. Med. 15(11):
501-509). Endothelial progenitor cells (EPCs), meaning cells that
exhibit clonal expression, stemness characteristics, adherence to
matrix molecules and an ability to differentiate into endothelial
cells (ECs) have been implicated in the formation of new blood
vessels through angiogenesis and postnatal vasculogenesis. EPCs
have many characteristic cell surface markers, including, but not
limited to, CD34, AC133, KDR (VEGFR-2), Tie-2 and ligand for UEA-1
lectin. (Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509;
Melero-Martin and Dudley, 2011, Stem Cells, 29: 163-168; Pascilli
et al., 2008, Exp. Cell Res., 315: 901-914).
EPC niches have been identified in the bone-marrow, peripheral cord
blood and vascular wall matrix. Bone-marrow derived and cord blood
EPCs essentially may be proangiogenic hematopoietic progenitor
cells (HPCs), circulating in the blood and committed to myeloid
lineage. (Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509).
The vascular wall stem and progenitor cells (VW-EPCs) reside in
distinct zones of the vessel wall within subendothelial space,
known as avasculogenic zone, within the vascular adventitia,
forming vascular wall-specific niches. Fetal and adult arterial and
venous blood vessel walls have also been found to harbor resident
niches for a variety of stem and progenitor cells, such as EPCs,
smooth muscle progenitors, HSCs, MSCs, mesangial cells coexpressing
myogenic and endothelial markers, neural stem cells (NSCs), etc.
(Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509). The VW-EPCs
are CD34(+)VEGFR-2(+)Tie-2(+)CD31(-)CD144(-). Proliferating and
differentiating VW-EPCs become CD144(+).
During embryogenesis, there is evidence of the existence of a
hemangioblast (giving rise to endothelial and hematopoietic cells)
and hemogenic endothelium, originating from precursors resident in
the vascular wall. However, whether adult VW also contains
ancestral progenitor hemangioblasts giving rise to both VW-EPCs as
well as VW-HSCs is not known. Vascular wall also contains resident
pericyte-like cells in the subendothelial spaces. These
pericyte-like cells serve as a cellular reservoir for VW-MSCs,
which can differentiate into colonies with adipogenic, osteogenic
and chondrogenic markers. (Tilki et al., 2009, Trends Mol. Med.
15(11): 501-509).
3. Cells of the Epithelial Tissue Compartment
3.1. Placental Tissue Matrix
The placenta is considered one of the most important sources of
stem cells, and has been studied extensively. It fulfills two main
desiderata of cell therapy: a source of a high as possible number
of cells and the use of non-invasive methods for their harvesting.
Their high immunological tolerance supports their use as an
adequate source in cell therapy (Mihu, C. et al., 2008, Romanian
Journal of Morphology and Embryology, 2008, 49(4):441-446).
The fetal adnexa is composed of the placenta, fetal membranes, and
umbilical cord. The term placenta is discoid in shape with a
diameter of 15-20 cm and a thickness of 2-3 cm. The fetal
membranes, amnion and chorion, which enclose the fetus in the
amniotic cavity, and the endometrial decidua extend from the
margins of the chorionic disc. The chorionic plate is a
multilayered structure that faces the amniotic cavity. It consists
of two different structures: the amniotic membrane (composed of
epithelium, compact layer, amniotic mesoderm, and spongy layer) and
the chorion (composed of mesenchyme and a region of extravillous
proliferating trophoblast cells interposed in varying amounts of
Langhans fibrinoid, either covered or not by
syncytiotrophoblast).
Villi originate from the chorionic plate and anchor the placenta
through the trophoblast of the basal plate and maternal
endometrium. From the maternal side, protrusions of the basal plate
within the chorionic villi produce the placental septa, which
divide the parenchyma into irregular cotyledons (Parolini, O. et
al., 2008, Stem Cell, 2008, 26:300-311).
Some villi anchor the placenta to the basal plate, whereas others
terminate freely in the intervillous space. Chorionic villi present
with different functions and structure. In the term placenta, the
stem villi show an inner core of fetal vessels with a distinct
muscular wall and connective tissue consisting of fibroblasts,
myofibroblasts, and dispersed tissue macrophages (Hofbauer cells).
Mature intermediate villi and term villi are composed of capillary
vessels and thin mesenchyme. A basement membrane separates the
stromal core from an uninterrupted multinucleated layer, called the
syncytiotrophoblast. Between the syncytiotrophoblast and its
basement membrane are single or aggregated Langhans
cytotrophoblastic cells, commonly called cytotrophoblast cells
(Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
Four regions of fetal placenta can be distinguished: an amniotic
epithelial region, an amniotic mesenchymal region, a chorionic
mesenchymal region, and a chorionic trophoblastic region.
Amniotic Membrane
Fetal membranes continue from the edge of the placenta and enclose
the amniotic fluid and the fetus. The amnion is a thin, avascular
membrane composed of an inner epithelial layer and an outer layer
of connective tissue that, and is contiguous, over the umbilical
cord, with the fetal skin. The amniotic epithelium (AE) is an
uninterrupted, single layer of flat, cuboidal and columnar
epithelial cells in contact with amniotic fluid. It is attached to
a distinct basal lamina that is, in turn, connected to the amniotic
mesoderm (AM). In the amniotic mesoderm closest to the epithelium,
an acellular compact layer is distinguishable, composed of
collagens I and III and fibronectin. Deeper in the AM, a network of
dispersed fibroblast-like mesenchymal cells and rare macrophages
are observed. It has been reported that the mesenchymal layer of
amnion indeed contains two subfractions, one having a mesenchymal
phenotype, also known as amniotic mesenchymal stromal cells, and
the second containing monocyte-like cells.
Chorionic Membrane
A spongy layer of loosely arranged collagen fibers separates the
amniotic and chorionic mesoderm. The chorionic membrane (chorion
laeve) consists of mesodermal and trophoblastic regions. Chorionic
and amniotic mesoderm are similar in composition. A large and
incomplete basal lamina separates the chorionic mesoderm from the
extravillous trophoblast cells. The latter, similar to trophoblast
cells present in the basal plate, are dispersed within the
fibrinoid layer and express immunohistochemical markers of
proliferation. The Langhans fibrinoid layer usually increases
during pregnancy and is composed of two different types of
fibrinoid: a matrix type on the inner side (more compact) and a
fibrin type on the outer side (more reticulate). At the edge of the
placenta and in the basal plate, the trophoblast interdigitates
extensively with the decidua (Cunningham, F. et al., The placenta
and fetal membranes, Williams Obstetrics, 20th ed. Appleton and
Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of
the human placenta. New York, Springer-Verlag, 2000, 42-46, 116,
281-297).
Amnion-Derived Stem Cells
The amniotic membrane itself contains multipotent cells that are
able to differentiate in the various layers. Studies have reported
their potential in neural and glial cells, cardiac repair and also
hepatocyte cells. Studies have shown that human amniotic epithelial
cells express stem cell markers and have the ability to
differentiate toward all three germ layers. These properties, the
ease of isolation of the cells, and the availability of placenta,
make amnionic membrane a useful and noncontroversial source of
cells for transplantation and regenerative medicine.
Amniotic epithelial cells can be isolated from the amniotic
membrane by several methods that are known in the art. According to
one such method, the aminiotic membrane is stripped from the
underlying chorion and digested with trypsin or other digestive
enzymes. The isolated cells readily attach to plastic or basement
membrane-coated culture dishes. Culture is established commonly in
a simple medium such as Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 5%-10% serum and epidermal growth factor (EGF),
in which the cells proliferate robustly and display typical
cuboidal epithelial morphology. Normally, 2-6 passages are possible
before proliferation ceases. Amniotic epithelial cells do not
proliferate well at low densities.
Amniotic membrane contains epithelial cells with different surface
markers, suggesting some heterogeneity of phenotype. Immediately
after isolation, human amniotic epithelial cells express very low
levels of human leukocyte antigen (HLA)-A, B, C; however, by
passage 2, significant levels are observed. Additional cell surface
antigens on human amniotic epithelial cells include, but are not
limited to, ATP-binding cassette transporter G2 (ABCG2/BCRP), CD9,
CD24, E-cadherin, integrins .alpha.6 and .beta.1, c-met (hepatocyte
growth factor receptor), stage-specific embryonic antigens (SSEAs)
3 and 4, and tumor rejection antigens 1-60 and 1-81. Surface
markers thought to be absent on human amniotic epithelial cells
include SSEA-1, CD34, and CD133, whereas other markers, such as
CD117 (c-kit) and CCR4 (CC chemokine receptor), are either negative
or may be expressed on some cells at very low levels. Although
initial cell isolates express very low levels of CD90 (Thy-1), the
expression of this antigen increases rapidly in culture (Miki, T.
et al., Stem Cells, 2005, 23: 1549-1559; Miki, T. et al., Stem
Cells, 2006, 2: 133-142).
In addition to surface markers, human amniotic epithelial cells
express molecular markers of pluripotent stem cells, including
octamer-binding protein 4 (OCT-4) SRY-related HMG-box gene 2
(SOX-2), and Nanog (Miki, T. et al., Stem Cells, 2005, 23:
1549-1559). Previous studies also have shown that human amnion
cells in xenogeneic, chimeric aggregates, which contain mouse
embryonic stem cells, can differentiate into all three germ layers
and that cultured human amniotic epithelial cells express neural
and glial markers, and can synthesize and release acetylcholine,
catecholamines, and dopamine. Hepatic differentiation of human
amniotic epithelial cells also has been reported. Studies have
reported that cultured human amniotic epithelial cells produce
albumin and .alpha.-fetroprotein and that albumin and
.alpha.-fetroprotein-positive hepatocyte-like cells could be
identified integrated into hepatic parenchyma following
transplantation of human amniotic epithelial cells into the livers
of severe combined immunodeficiency (SCID) mice. The hepatic
potential of human amniotic epithelial cells was confirmed and
extended, whereby in addition to albumin and .alpha.-fetroprotein
production, other hepatic functions, such as glycogen storage and
expression of liver-enriched transcription factors, such as
hepatocyte nuclear factor (HNF) 3.gamma. and HNF4.alpha.,
CCAAT/enhancer-binding protein (CEBP .alpha. and .beta.), and
several of the drug metabolizing genes (cytochrome P450) were
demonstrated. The wide range of hepatic genes and functions
identified in human amniotic epithelial cells has suggested that
these cells may be useful for liver-directed cell therapy
(Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).
Differentiation of human amniotic epithelial cells to another
endodermal tissue, pancreas, also has been reported. For example,
it was shown that human amniotic epithelial cells cultured for 2-4
weeks in the presence of nicotinamide to induce pancreatic
differentiation, expressed insulin. Subsequent transplantation of
the insulin-expressing human amniotic epithelial cells corrected
the hyperglycemia of streptozotocin-induced diabetic mice. In the
same setting, human amniotic mesenchymal stromal cells were
ineffective, suggesting that human amniotic epithelial cells, but
not human amniotic mesenchymal stromal cells, were capable of
acquiring .beta.-cell fate (Parolini, O. et al., 2008, Stem Cell,
2008, 26:300-311).
Mesenchymal Stromal Cells from Amnion and Chorion: hAMSC and
hCMSC
Human amniotic mesenchymal cells (hAMSC) and human chorionic
mesenchymal cells (hCMSC) are thought to be derived from
extraembryonic mesoderm. hAMSC and hCMSC can be isolated from
first-, second-, and third-trimester mesoderm of amnion and
chorion, respectively. For hAMSC, isolations are usually performed
with term amnion dissected from the deflected part of the fetal
membranes to minimize the presence of maternal cells. For example,
homogenous hAMSC populations can be obtained by a two-step
procedure, whereby: minced amnion tissue is treated with trypsin to
remove hAEC and the remaining mesenchymal cells are then released
by digestion (e.g., with collagenase or collagenase and DNase). The
yield from term amnion is about 1 million hAMSC and 10-fold more
hAEC per gram of tissue (Casey, M. and MacDonald P., Biol Reprod,
1996, 55: 1253-1260).
hCMSCs are isolated from both first- and third-trimester chorion
after mechanical and enzymatic removal of the trophoblastic layer
with dispase. Chorionic mesodermal tissue is then digested (e.g.,
with collagenase or collagenase plus DNase). Mesenchymal cells also
have been isolated from chorionic fetal villi through explant
culture, although maternal contamination is more likely (Zhang, X.,
et al., Biochem Biophys Res Commun, 2006, 340: 944-952; Soncini, M.
et al., J Tissue Eng Regen Med, 2007, 1:296-305; Zhang et al.,
Biochem Biophys Res Commun, 2006, 351: 853-859).
The surface marker profile of cultured hAMSC and hCMSC, and
mesenchymal stromal cells (MSC) from adult bone marrow are similar.
All express typical mesenchymal markers (Table 7) but are negative
for hematopoietic (CD34 and CD45) and monocytic markers (CD14).
Surface expression of SSEA-3 and SSEA-4 and RNA for OCT-4 has been
reported (Wei J. et al., Cell Transplant, 2003, 12: 545-552;
Wolbank, S. et al., Tissue Eng, 2007, 13: 1173-1183; Alviano, F. et
al., BMC Dev Biol, 2007, 7: 11; Zhao, P. et al, Transplantation,
2005, 79: 528-535). Both first- and third trimester hAMSC and hCMSC
express low levels of HLA-A, B, C but not HLA-DR, indicating an
immunoprivileged status (Portmann-Lanz, C. et al, Am J Obstet
Gynecol, 2006, 194: 664-673; Wolbank, S. et al., Tissue Eng, 2007,
13: 1173-1183).
Table 10 provides surface antigen expression profile at passages
2-4 for amniotic mesenchymal stromal and human chorionic
mesenchymal stromal stem cells.
TABLE-US-00010 TABLE 10 Specific surface antigen expression for
aminiotic mesenchymal stromal cells and human chorionic mesenchymal
stromal cells Positive (.gtoreq.95%) Negative (.ltoreq.2%) CD90
CD45 CD73 CD34 CD105 HLA-DR
Both hAMSCs and hCMSCs differentiate toward "classic" mesodermal
lineages (osteogenic, chondrogenic, and adipogenic) and
differentiation of hAMSC to all three germ layers-ectoderm
(neural), mesoderm (skeletal muscle, cardiomyocytic and
endothelial), and endoderm (pancreatic) was reported (Int'Anker, P.
et al., Stem Cells, 2004, 22: 1338-1345; Portmann-Lanz, C. et al,
Am J Obstet Gynecol, 2006, 194: 664-673; Wolbank, S. et al., Tissue
Eng, 2007, 13: 1173-1183; Soncini, M. et al., J Tissue Eng Regen
Med, 2007, 1:296-305; Alviano, F., BMC Dev Biol, 2007, 7: 11).
Human amniotic and chorionic cells successfully and persistently
engraft in multiple organs and tissues in vivo. Human chimerism
detection in brain, lung, bone marrow, thymus, spleen, kidney, and
liver after either intraperitoneal or intravenous transplantation
of human amnion and chorion cells into neonatal swine and rats was
indeed indicative of an active migration consistent with the
expression of adhesion and migration molecules (L-selectin, VLA-5,
CD29, and P-selectin ligand 1), as well as cellular matrix
proteinase (MMP-2 and MMP-9) (Bailo, M. et al., Transplantation,
2004, 78:1439-1448).
Umbilical Cord
Two types of umbilical stem cells can be found, namely
hematopoietic stem cells (UC-HS) and mesenchymal stem cells, which
in turn can be found in umbilical cord blood (UC-MS) or in
Wharton's jelly (UC-MM). The blood of the umbilical cord has long
been in the focus of attention of researchers as an important
source of stem cells for transplantation, for several reasons: (1)
it contains a higher number of primitive hematopoietic stem cells
(HSC) per volume unit, which proliferate more rapidly, than bone
marrow; (2) there is a lower risk of rejection after
transplantation; (3) transplantation does not require a perfect HLA
antigen match (unlike in the case of bone marrow); (4) UC blood has
already been successfully used in the treatment of inborn metabolic
errors; and (5) there is no need for a new technology for
collection and storage of the mononuclear cells from UC blood,
since such methods are long established.
Umbilical cord (UC) vessels and the surrounding mesenchyma
(including the connective tissue known as Wharton's jelly) derive
from the embryonic and/or extraembryonic mesodermis. Thus, these
tissues, as well as the primitive germ cells, are differentiated
from the proximal epiblast, at the time of formation of the
primitive line of the embryo, containing MSC and even some cells
with pluripotent potential. The UC matrix material is speculated to
be derived from a primitive mesenchyma, which is in a transition
state towards the adult bone marrow mesenchyma (Mihu, C. et al.,
2008, Romanian Journal of Morphology and Embryology, 2008,
49(4):441-446).
The blood from the placenta and the umbilical cord is relatively
easy to collect in usual blood donation bags, which contain
anticoagulant substances. Mononuclear cells are separated by
centrifugation on Ficoll gradient, from which the two stem cell
populations will be separated: (1) hematopoietic stem cells (HSC),
which express certain characteristic markers (CD34, CD133); and (2)
mesenchymal stem cells (MSC) that adhere to the culture surface
under certain conditions (e.g., modified McCoy medium and lining of
vessels with Fetal Bovine Serum (FBS) or Fetal Calf Serum (FCS)).
(Munn, D. et al., Science, 1998, 281: 1191-1193; Munn, D. et al., J
Exp Med, 1999, 189: 1363-1372). Umbilical cord blood MSCs (UC-MS)
can produce cytokines, which facilitate grafting in the donor and
in vitro HSC survival compared to bone marrow MSC. (Zhang, X et
al., Biochem Biophys Res Commun, 2006, 351: 853-859).
MSCs from the umbilical cord matrix (UC-MM) are obtained by
different culture methods depending on the source of cells, e.g.,
MSCs from the connective matrix, from subendothelial cells from the
umbilical vein or even from whole umbilical cord explant. They are
generally well cultured in DMEM medium, supplemented with various
nutritional and growth factors; in certain cases prior treatment of
vessels with hyaluronic acid has proved beneficial (Baban, B. et
al., J Reprod Immunol, 2004, 61: 67-77).
3.2. Lung
The lungs, which are paired organs that fill up the thoracic
cavity, constitute an efficient air-blood gaseous exchange
mechanism, accomplished by the passage of air from the mouth or
nose, sequentially through an oropharynx, nasopharynx, a larynx, a
trachea and finally through a progressively subdividing system of
bronchi and bronchioles until it finally reaches alveoli where the
air-blood gaseous exchange takes place. A resident niche with
characteristic multipotent stem cells with c-kit positive surface
profiles recently has been identified localized in small
bronchioles alveoli. These stem cells express the transcription
factors, Nanog, Oct3/4, Sox2 and Klf4, that govern pluripotency in
embryonic stem cells. (Kajstura, J. et al., 2011, New Engl. J.
Med., 364(19):1795-1806)).
3.3. Mammary
The mammary gland is a hormone sensitive bilayered epithelial organ
comprising an inner luminal epithelial layer and an outer
myoepithelial layer surrounded by a basement membrane in a stromal
fat pad. Mammary stem cells with myoepithelial potential have been
identified in their niches in the terminal ducts of mammary gland.
(LaBarge, 2007, Stem Cell Rev., 3(2): 137-146).
3.4. Skin
The skin functions as the primary barrier imparting protection from
environmental insults. Skin is composed of an outer epidermis and
inner dermis separated by a basement membrane (BM), rich in ECM and
growth factors. The BM of the epidermal-dermal junction is composed
of collagens (e.g., type IV and XVII), laminins, nidogen,
fibronectin and proteoglycans that provide storage sites for growth
factors and nutrients supporting the proliferation and adhesion of
epidermal keratinocytes.
The epidermis is a solid epithelial tissue comprising keratinocytes
that are linked to each other via cellular junctions, such as
desmosomes. Keratinocytes are organized into distinct layers,
comprising the stratum corneum, stratum granulosum, stratum
spinosum and stratum basale. The epidermal matrix is made up of
hyaluronan and other proteoglycans, including but not limited to,
desmosealin, glycipans, versican, perlecan, and syndecans. (Sandjeu
and Haftek, 2009, J. Physiol. Pharmacol. 60 (S4): 23-30). Epidermal
desmosomes are multimeric complexes of transmembrane glycoprotein
and cytosolic proteins with the keratin cytoskeleton. Desmosal
proteins of the epidermis predominantly belong to the cadherin,
Armadillo and plakin superfamilies.
The underlying dermis is connective tissue comprised primarily of
fibroblasts with occasional inflammatory cells. Embedded within the
dermis are also epidermal appendages, such as hair follicles and
sebaceous glands, as well as nerves and cutaneous vasculature. The
dermal ECM is essentially made of type I, III and V collagens and
elastin together with noncollagenous components such as
glycoproteins, proteoglycans, GAGs, cytokines and growth factors.
Dermal collagens help mediate fibroblast-matrix interactions
through a number of cell surface receptors and proteoglycans, such
as .beta.1-integrins. (Hodde and Johnson, 2007, Am. J. Clin.
Dermatol. 8(2): 61-66).
During embryonic development, the epidermis originates from the
ectoderm, while the dermis differentiates from the mesoderm.
Following gastrulation, as mesenchymal stem cells of mesodermal
origin populate the skin, they send signals to the single epidermal
layer for initiation of epidermal stratification and direct the
positioning of outgrowths of epidermal appendages, such as the hair
follicles and sebaceous glands. Along with the mesenchyme, the
basal layer of the epidermis organizes into a basement membrane
that is rich in ECM proteins and growth factors. A number of
different signaling pathways have been implicated in skin
morphogenesis, including but not limited to Notch, Wnt, mitogen
activated protein kinase (MAPK), nuclear factor-.kappa.B
(NF-.kappa.B), transcriptional regulator, p63, the AP2 family of
transcription factors, CCAAT/enhancer binding protein (C/EBP)
transcriptional regulators, interferon regulatory 6 (URF6),
grainyhead-like 3 (GRHL3) and Kruppel-like factor (KLF4). (Blanpain
and Fuchs, 2009, Nat. Rev. Mol. Cell. Biol., 10(3): 207-217).
Adult skin undergoes constant cellular turnover whereby dead skin
cells are shed and new cells are regenerated and replaced, by a
process known as skin homeostasis. Several stem cell niches with
distinct surface marker profiles and differentiation potentials
have been identified. These include, but are not limited to,
epidermal stem cells of interfollicular epidermis, bulge stem cells
and epithelial stem cells of the hair follicle, dermal stem cells
(e.g., multipotent dermal cells, skin-derived progenitor cells,
dermis-derived multipotent stem cells and fibrocytes), dermal
papilla stem cells, and sebaceous gland stem cells. Collectively,
these skin stem cell niches partake in maintaining skin homeostasis
with the help of growth factors and cytokines. (Zouboulis et al.,
2008, Exp. Gerontol. 43: 986-997; Blanpain, 2010, Nature, 464:
686-687).
4. Cells of the Muscular Tissue Compartment
The muscular tissue compartments are comprised of contractile
muscle tissue. These can be of three kinds: skeletal muscle
associated with the skeletal system; cardiac muscle associated with
the heart; and smooth muscle associated with the vasculature and
gastrointestinal tract. Skeletal muscle tissue fibers are striated
and are voluntary in function. Cardiac muscle fibers have
characteristic intercalated discs and are involuntary in function.
Smooth muscle tissue is comprised of spindle shaped cells and is
involuntary in function.
Skeletal muscles are composed of a population of quiescent myogenic
precursor cells known as satellite cells with muscle regenerating
and self-renewal properties, as well as a population of multipotent
muscle-derived stem cells (MDSC) with multilineage differentiation
potential, such as mesodermal lineages including, but not limited
to, myogenic lineages, adipogenic lineages, osteogenic lineages,
chondrogenic lineages, endothelial and hematopoetic lineages, and
ectodermal lineages, including not limited to neuron-like cells.
(Xu et al., 2010, Cell Tissue Res., 340: 549-567).
Skeletal muscle satellite cells are quiescent mononucleated cells
that are resident in the muscle fiber membrane, beneath the basal
lamina forming distinct stem cell niches. Similar to other stem
cell niches, the skeletal muscle satellite cell niche is a dynamic
structure, capable of altering between inactive (quiescent) and
activated states in response to external signals. Once activated,
satellite cells have the potential to proliferate, expand and
differentiate along the myogenic lineage. The basal lamina, which
serves to separate individual skeletal muscle fibers, known as
myofibers, and their associated satellite cell and stem cell
niches, from the cells of the interstitium, is rich in collagen
type IV, perlecan, laminin, entactin, fibronectin and several other
glycoproteins and proteoglycans, that may function as receptors to
growth factors effectuating their activation by extracellular
processing and modifications. In addition to these interactions
provided by the ECM, neighboring cells, such as endothelial cells
and multipotent stem cells derived from blood vessels, such as
pericytes and mesoangioblasts, or neural components, all have the
potential of affecting the niche microenvironment. (Gopinath et
al., 2008, Aging Cell, 7: 590-598).
Endogenous cardiac stem cells have also been identified in cardiac
stem cell niches. (Mazhari and Hare, 2007, Nat. Clin. Pract.
Cardiovasc. Med., 4(S1): S21-S26).
Vascular smooth muscle cells are derived from embryonic cardiac
neural crest stem cells, as well as proepicardial cells and
endothelial progenitor cells. Smooth muscle differentiation is
dependent on a combination of factors, including but not limited to
Pax3, Tbx1, FoxC1 and serum response factor, interacting with
microenvironment components of the ECM, such as BMPs, Wnts,
endothelin (ET)-1, and FGF8. In the adult, vascular smooth muscle
cells undergo constant degeneration, repair and regeneration by the
concerted efforts of both multipotent bone-derived mesenchymal
cells as well as smooth muscle stem cells resident within vascular
smooth muscle tissue. (Hirschi and Majesky, 2004, The Anatomical
Record, Part A, 276A: 22-33).
5. Cells of the Neural Tissue Compartment
The neural tissue compartments are comprised of neurons and the
neuroglia, embedded with the neural matrix. Neural tissue is
ectodermal in origin, derived from the embryonic neural plate.
Neural tissue is primarily located within the brain, spinal cord
and nerves.
Resident neural stem cell niches have been identified in the adult
mammalian brain, restricted to the subventricular zone as well as
to the lateral ventricle and dentate gyrus subgranular zone of the
hippocampus. Astrocytes, which are star-shaped nerve cells, serve
as both neural stem cells as well as supporting niche cells
secreting essential growth factors that provide support for
neurogenesis and vasculogenesis. The basal lamina and associated
vasculogenesis are essential components of the niche. Embryonic
molecular factors and signals persist within the neural stem cell
niches and play critical role in neurogenesis. Neural stem cells
have VEGFR2, doublecortin and Lex (CD15) markers. Major signaling
pathways implicated in neurogenesis include but are not limited to
Notch, Eph/ephrins, Shh, and BMPs. (Alvarez-Buylla and Lim, 2004,
Neuron, 41: 683-686).
6. Grafts--Grafts and Graft Rejection
A graft is a tissue or organ used for transplantation to a patient.
A common strategy employed in tissue engineering involves the
seeding of decellularized natural ECM or synthetic scaffolds with a
variety of different stem or progenitor cells that are capable of
regeneration (see, for example, Flynn and Woodhouse, 2008,
Organogenesis, 4(4): 228-235; Uriel et al., 2008, Biomaterials, 29:
3712-3719; Flynn, 2010, Biomaterials, 31: 4715-4724; Choi et al.,
Tissue Engg. C., 16(3): 387-396; Brown et al., 2011, Tissue Engg.
C., 17(4): 411-421; Cheng et al., 2009, Tissue Engg. A, 15(2):
231-241; Li et al., 2011, Biomaterials,
doi:10:1016/j.biomaterials.2011.03.008; Butler et al., 2003,
Connective Tissue Research, 44(S1): 171-178); Mercuri et al., J.
Biomed. Mater. Res. A., 96(2): 422-435); Olson et al., 2011,
Chonnam. Med. J. 47:1-13).
Transplanted grafts may be rejected by the recipient host via an
orchestrated immune response against the histocompatibility
antigens expressed by the grafted tissue, which the recipient host
may see as foreign. Effectors primarily responsible for such
rejections include type 1 helper CD4+ cells, cytotoxic CD8+ cells
and antibodies. Alternative mechanisms of rejection include the
involvement of type 2 helper CD4+ cells, memory CD8+ cells, and
cells that belong to the innate immune system, such as natural
killer cells, eosinophils, and neutrophils. In addition, local
inflammation associated with rejection is tightly regulated at the
graft level by regulatory T cells and mast cells.
Implants
Patients suffering from affected or injured organs may be treated
with organ transplantation. However, current methods of organ
transplantation are faced with challenges due, in part to the need
to suppress immune rejection of the transplanted organ. Most
methods rely on the use of immunosuppressive drugs that are
associated with unwanted side effects.
It is estimated that more than one million patients need to be
treated surgically for skeletal afflictions every year due to bony
defects created during tumor surgery or caused by trauma,
congenital skeletal abnormalities, fracture, scoliosis, spinal
arthrodesis, or joint and tooth replacement. Surgical treatments,
however, are not always effective to address these problems because
of inadequate local bone conditions and impaired bone healing. For
example, complicated fractures may fail to heal, resulting in
delayed unions (a bone fracture that is taking an exceptionally
long amount of time to heal) or non-unions (absence of healing in a
fracture). In addition, the treatment of bone tumors or congenital
syndromes often requires the artificial creation of large bony
defects, which need to be filled, demanding suitable and
biocompatible substitutes for bone grafts.
Bone healing around implants involves the activation of a sequence
of osteogenic, vascular, and immunological events that are similar
to those occurring during bone healing. Various cell types, growth
factors and cytokines are involved and interact throughout the
stages of osteointegration, including inflammation,
vascularization, bone formation, and ultimately bone
remodeling.
Bone Grafts
Fresh autologous bone grafts for the treatment of an osseous defect
or fracture in a patient are derived from bone marrow freshly
harvested from the iliac crest (the thick curved upper border of
the ilium, the most prominent bone on the pelvis) and combined with
other materials including osteoconductive substrates. Complications
associated with autologous harvest include donor site morbidity as
high as 25%, infection, malformation, pain, and loss of
function.
Bone Matrix with Mesenchymal Stem Cells
Attempts have been made to repair osseous defects by implanting a
bone matrix comprising autologous or allogeneic mesenchymal stem
cells (MSCs). MSCs are considered immunologically neutral, meaning
that the mesenchymal stem cells from the donor need not be
tissue-matched to the recipient, thus allowing MSCs to be used
effectively in allogeneic grafts. In addition, culture-expanded
allogeneic MSCs have been implanted either directly or combined
with a matrix, such as a gelatin-based or collagen-based matrix, or
a bone matrix, in order to support differentiation of the MSCs in
vivo.
In other instances, MSCs have been combined with a bone matrix from
which bone marrow has been removed in order to remove undesirable
cells, and the matrix then seeded with culture-expanded MSCs. Such
compositions then are cryopreserved under standard cryopreservation
procedures for later use. However, this method is not ideal for
several reasons. First, because the MSCs have been removed from the
original stem cell niche and seeded onto a new bone matrix, the
MSCs in such a composition are not well-attached to the bone matrix
and become merely suspended in the cryopreservation solution. As a
result, many active cells can be lost during the process of
removing the cryopreservation solution before transplantation into
a subject. Secondly, since the cells are not attached to the stem
cell niche or lacunae to which they were originally attached and in
which they were nurtured, the expandability and osteogenic
potential of the cells may be affected negatively by the separation
and seeding procedures.
Tissue-derived implant materials replicate the biological and
mechanical function of naturally occurring extracellular matrix
found in body tissues. Such tissue-derived matrices provide the
necessary support on which cells can adhere to, migrate and expand
and allow the influx and efflux of cells, such as stem cells and
progenitor cells, and other factors, such as growth factors and
cytokines, capable of inducing and supporting growth and tissue
repair.
Glossary
The term "ambient temperature" as used herein refers to the
temperature of the immediate, unaltered surroundings. Ambient
temperature is between about 18.degree. C. and about 28.degree. C.
According to some embodiments, ambient temperature is room
temperature.
The term "adherent" as used herein refers to the act of sticking
to, clinging, or staying attached.
The term "adipokine" as used herein refers to a factor secreted by
adipose tissue.
The term "adipocyte" as used herein refers to the functional cell
type of fat, or adipose tissue, that is found throughout the body,
particularly under the skin. Adipocytes store and synthesize fat
(more specifically, triglycerides or lipids) for energy, thermal
regulation and cushioning against mechanical shock. Although the
lineage of adipocytes is still unclear, it appears that MSCs can
differentiate into two types of lipoblasts, one that give rise to
white adipocytes and the other to brown adipocytes. Both types of
adipocytes store triglycerides and other lipids.
The term "adipogenic" as used herein refers to a potential of
undifferentiated precursor cells to differentiate into fat-forming
or adipocompetent cells.
The term "adipose stem cell" or "adipose-derived stem cell" (ASC)
as used herein refers to pluripotent stem cells, MSCs and more
committed adipose progenitors and stroma obtained from adipose
tissue.
The term "administer" as used herein means to give or to apply.
The term "allogeneic" as used herein refers to being genetically
different although belonging to or obtained from the same
species.
The term "amniotic stem cells" as used herein refers to pluripotent
stem cells, multipotent stem cells and progenitor cells derived
from amniotic membrane, which can give rise to a limited number of
cell types in vitro and/or in vivo under an appropriate condition,
and expressly includes both amniotic epithelial cells and amniotic
stromal cells.
The term "attached" as used herein refers to being fastened, fixed,
joined, connected, bound, adhered to or assembled with.
The term "autologous" as used herein means derived from the same
organism.
The term "autologous graft" or "autograft" as used herein refers to
a tissue that is grafted into a new position in or on the body of
the same individual.
The term "basic fibroblast growth factor" (bFGF, FGF2) as used
herein refers to a multifunctional effector for many cells of
mesenchymal and neuroectodermal origin that is a potent inducer of
neovascularization and angiogenesis.
The term "biocompatible" as used herein refers to causing no
clinically relevant tissue irritation, injury, toxic reaction, or
immunological reaction to living tissue.
The term "biomarkers" (or "biosignatures") as used herein refers to
peptides, proteins, nucleic acids, antibodies, genes, metabolites,
or any other substances used as indicators of a biologic state. It
is a characteristic that is measured objectively and evaluated as a
cellular or molecular indicator of normal biologic processes,
pathogenic processes, or pharmacologic responses to a therapeutic
intervention.
The term "bone" as used herein refers to a hard connective tissue
consisting of cells embedded in a matrix of mineralized ground
substance and collagen fibers. The fibers are impregnated with a
form of calcium phosphate similar to hydroxyapatite as well as with
substantial quantities of carbonate, citrate and magnesium. Bone
consists of a dense outer layer of compact substance or cortical
substance covered by the periosteum and an inner loose, spongy
substance; the central portion of a long bone is filled with
marrow.
The terms "cancellous bone" or "trabecular bone" as used herein
refer to the spongy bone found in the inner parts of compact bone
in which the matrix forms a lattice of large plates and rods known
as the trabeculae, which anastomose to form a latticework. This
latticework partially encloses many intercommunicating spaces
filled with bone marrow. The marrow spaces are relatively large and
irregularly arranged, and the bone substance is in the form of
slender anastomosing trabeculae and pointed spicules.
The terms "cortical bone" or "compact bone" as used herein refer to
the dense outer layer of bone that consists largely of concentric
lamellar osteons and interstitial lamellae. The spaces or channels
are narrow and the bone substance is densely packed.
The term "bone morphogenetic protein (BMP) as used herein refers to
a group of cytokines that are part of the transforming growth
factor- (TGF- ) superfamily. BMP ligands bind to a complex of the
BMP receptor type II and a BMP receptor type I (Ia or Ib). This
leads to the phosphorylation of the type I receptor that
subsequently phosphorylates the BMP-specific Smads (Smad1, Smad5,
and Smad8), allowing these receptor-associated Smads to form a
complex with Smad4 and move into the nucleus where the Smad complex
binds a DNA binding protein and acts as a transcriptional enhancer.
BMPs have a significant role in bone and cartilage formation in
vivo. It has been reported that most BMPs are able to stimulate
osteogenesis in mature osteoblasts, while BMP-2, 6, and 9 may play
an important role in inducing osteoblast differentiation of
mesenchymal stem cells. Cheng, H. et al., J. Bone & Joint
Surgery 85: 1544-52 (2003).
The term "bound" or any of its grammatical forms as used herein
refers to the capacity to hold onto, attract, interact with or
combine with.
The term "buffer" or "buffer solution" as used herein refers to a
compound, usually a salt, which, when dissolved in an aqueous
medium, serves to maintain the free hydrogen ion concentration of
the solution within a certain pH range when hydrogen ions are added
or removed from the solution. A salt or solution is said to have a
"buffering capacity" or to buffer the solution over such a range,
when it provides this function. Generally a buffer will have
adequate buffering capacity over a range that is within ..+-..1 pH
unit of its pK.
The term "buffered isotonic solution" as used herein refers to any
buffer that is commonly used in biological research. Exemplary
buffered isotonic solutions include but are not limited to balanced
salt solution (BSS), Hank's Balanced Salt Solution, Gey's Balanced
Salt Solution, Hank's Buffered Salt Solution, Phosphate Buffered
Saline, Tris-Buffered Saline, etc. The term "isotonic solution" as
used herein refers to a solution whose osmolarity and ion
concentrations closely match those within normal cells of the body
and the blood.
The term "carrier" as used herein refer to a pharmaceutically
acceptable inert agent or vehicle for delivering one or more active
agents to a subject, and often is referred to as "excipient." The
carrier must be of sufficiently high purity and of sufficiently low
toxicity to render it suitable for administration to the subject
being treated. The carrier further should maintain the stability
and bioavailability of an active agent
The term "cell" is used herein to refer to the structural and
functional unit of living organisms and is the smallest unit of an
organism classified as living.
The term "chemokine" as used herein refers to a class of
chemotactic cytokines that signal leukocytes to move in a specific
direction.
The terms "chemotaxis" or "chemotactic" refer to the directed
motion of a motile cell or part along a chemical concentration
gradient towards environmental conditions it deems attractive
and/or away from surroundings it finds repellent.
The term "chondrocytes" as used herein refers to cells found in
cartilage that produce and maintain the cartilaginous matrix for,
for example, joints, ear canals, trachea, epiglottis, larynx, the
discs between vertebrae and the ends of ribs. From least to
terminally differentiated, the chondrocytic lineage is (i)
Colony-forming unit-fibroblast (CFU-F); (ii) mesenchymal stem
cell/marrow stromal cell (MSC); (iii) chondrocyte.
The term "chondrogenesis" as used herein refers to the formation of
new cartilage from cartilage forming or chondrocompetent cells.
The term "chondrogenic" as used herein refers to a potential of
undifferentiated precursor cells to differentiate into cartilage
forming or chondrocompetent cells.
The term "compatible" as used herein means that the components of a
composition are capable of being combined with each other in a
manner such that there is no interaction that would substantially
reduce the efficacy of the composition under ordinary use
conditions.
The term "component" as used herein refers to a constituent part,
element or ingredient.
The term "condition", as used herein, refers to a variety of health
states and is meant to include disorders or diseases caused by any
underlying mechanism or disorder, injury, and the promotion of
healthy tissues and organs.
The term "contact" and its various grammatical forms as used herein
refers to a state or condition of touching or of immediate or local
proximity. Contacting a composition to a target destination may
occur by any means of administration known to the skilled
artisan.
The term "cut section thickness" as used herein refers to thickness
of a section as measured directly from the sectioning device
(cryostat, microtome, etc.) prior to histological processing, which
may cause shrinkage in the z-axis. Also known as the block advance
of the microtome.
The term "cytokine" as used herein refers to small soluble protein
substances secreted by cells which have a variety of effects on
other cells. Cytokines mediate many important physiological
functions including growth, development, wound healing, and the
immune response. They act by binding to their cell-specific
receptors located in the cell membrane, which allows a distinct
signal transduction cascade to start in the cell, which eventually
will lead to biochemical and phenotypic changes in target cells.
Generally, cytokines act locally. They include type I cytokines,
which encompass many of the interleukins, as well as several
hematopoietic growth factors; type II cytokines, including the
interferons and interleukin-10; tumor necrosis factor
("TNF")-related molecules, including TNF.alpha. and lymphotoxin;
immunoglobulin super-family members, including interleukin 1
("IL-1"); and the chemokines, a family of molecules that play a
critical role in a wide variety of immune and inflammatory
functions. The same cytokine can have different effects on a cell
depending on the state of the cell. Cytokines often regulate the
expression of, and trigger cascades of other cytokines. Nonlimiting
examples of cytokines include e.g., IL-1.alpha., IL-.beta., IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12/IL-23
P40, IL13, IL-17, IL-18, TGF-beta., IFN-gamma., GM-CSF, Gro.alpha.,
MCP-1 and TNF-alpha.
The term "cytometry" as used herein refers to a process in which
physical and/or chemical characteristics of single cells, or by
extension, of other biological or nonbiological particles in
roughly the same size or stage, are measured. In flow cytometry,
the measurements are made as the cells or particles pass through
the measuring apparatus (a flow cytometer) in a fluid stream. A
cell sorter, or flow sorter, is a flow cytometer that uses
electrical and/or mechanical means to divert and collect cells (or
other small particles) with measured characteristics that fall
within a user-selected range of values.
"Decellularization", as used herein in all of its grammatical
forms, is any process by which cells and cellular components
(including DNA) are removed from a tissue, thereby leaving the
extracellular matrix (ECM) essentially free of such cells and
cellular components.
"Delipidization" and "delipidation", as used herein in all of their
grammatical forms, are any processes by which lipids are removed
from a tissue, and are used interchangeably herein.
"Demineralized bone matrix" (DBM) refers to a bone-derived material
that has osteoconductive and osteoinductive activity. DBM may be
prepared by acid extraction of allograft bone, resulting in loss of
most of the mineralized component but retention of collagen and
noncollagenous proteins, including growth factors. Methods for
preparing demineralized bone matrix from bone are known in the art,
as disclosed, for example, in U.S. Pat. Nos. 5,073,373; 5,484,601;
and 5,284,655, which are incorporated herein by reference. DBM may
be prepared from autologous bone, allogeneic (or "allograft") bone,
or xenogeneic bone. DBM may be prepared from cancellous bone,
cortical bone, or combinations of cancellous and cortical bone. For
the purpose of the present disclosure, demineralized bone includes
bone matrix having a residual mineral content of 5% or less (w/w),
2% or less (w/w), 1% or less (w/w), 0.5% or less (w/w), or
consisting essentially of collagen, non-collagen proteins such as
growth factors, and other nonmineral substances found in the
original bone, although not necessarily in their original
quantities. The term "demineralized cortical bone" (DCB) as used
herein refers to a demineralized allograft cortical bone.
The term "derivative" as used herein means a compound that may be
produced from another compound of similar structure in one or more
steps. A "derivative" or "derivatives" of a peptide or a compound
retains at least a degree of the desired function of the peptide or
compound. Accordingly, an alternate term for "derivative" may be
"functional derivative." Derivatives can include chemical
modifications of the peptide, such as akylation, acylation,
carbamylation, iodination or any modification that derivatizes the
peptide. Such derivatized molecules include, for example, those
molecules in which free amino groups have been derivatized to form
amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formal
groups. Free carboxyl groups can be derivatized to form salts,
esters, amides, or hydrazides. Free hydroxyl groups can be
derivatized to form O-acyl or O-alkyl derivatives. The imidazole
nitrogen of histidine can be derivatized to form
N-im-benzylhistidine. Also included as derivatives or analogues are
those peptides that contain one or more naturally occurring amino
acid derivative of the twenty standard amino acids, for example,
4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,
ornithine or carboxyglutamiate, and can include amino acids that
are not linked by peptide bonds. Such peptide derivatives can be
incorporated during synthesis of a peptide, or a peptide can be
modified by well-known chemical modification methods (see, e.g.,
Glazer et al., Chemical Modification of Proteins, Selected Methods
and Analytical Procedures, Elsevier Biomedical Press, New York
(1975)).
The term "detectable marker" encompasses both selectable markers
and assay markers. The term "selectable markers" refers to a
variety of gene products to which cells transformed with an
expression construct can be selected or screened, including
drug-resistance markers, antigenic markers useful in
fluorescence-activated cell sorting, adherence markers such as
receptors for adherence ligands allowing selective adherence, and
the like.
The term "detectable response" refers to any signal or response
that may be detected in an assay, which may be performed with or
without a detection reagent. Detectable responses include, but are
not limited to, radioactive decay and energy (e.g., fluorescent,
ultraviolet, infrared, visible) emission, absorption, polarization,
fluorescence, phosphorescence, transmission, reflection or
resonance transfer. Detectable responses also include
chromatographic mobility, turbidity, electrophoretic mobility, mass
spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic
resonance spectrum and x-ray diffraction. Alternatively, a
detectable response may be the result of an assay to measure one or
more properties of a biologic material, such as melting point,
density, conductivity, surface acoustic waves, catalytic activity
or elemental composition. A "detection reagent" is any molecule
that generates a detectable response indicative of the presence or
absence of a substance of interest. Detection reagents include any
of a variety of molecules, such as antibodies, nucleic acid
sequences and enzymes. To facilitate detection, a detection reagent
may comprise a marker.
The term "devitalization", as used herein in all of its grammatical
forms, is any process which renders a tissue substantially or
essentially free from reproductively or metabolically viable cells,
without necessarily leaving the tissue essentially free of such
cells and cellular components.
The term "differential label" as used herein generally refers to a
stain, dye, marker, or antibody used to characterize or contrast
structures, components or proteins of a single cell or
organism.
The term "differentiation" as used herein refers to the process of
development with an increase in the level of organization or
complexity of a cell or tissue, accompanied with a more specialized
function.
The terms "disease" or "disorder" as used herein refer to an
impairment of health or a condition of abnormal functioning.
The term "disinfection", as used herein in all of its grammatical
forms, is any process which renders a tissue essentially free of
viable pathogenic organisms and viruses by destroying them or
otherwise inhibiting their growth or vital activity.
The term "dye" (also referred to as "fluorochrome" or
"fluorophore") as used herein refers to a component of a molecule
which causes the molecule to be fluorescent. The component is a
functional group in the molecule that absorbs energy of a specific
wavelength and re-emits energy at a different (but equally
specific) wavelength. The amount and wavelength of the emitted
energy depend on both the dye and the chemical environment of the
dye. Many dyes are known, including, but not limited to, FITC,
R-phycoerythrin (PE), PE-Texas Red Tandem, PE-Cy5 Tandem, propidium
iodem, EGFP, EYGP, ECF, DsRed, allophycocyanin (APC), PerCp, SYTOX
Green, courmarin, Alexa Fluors (350, 430, 488, 532, 546, 555, 568,
594, 633, 647, 660, 680, 700, 750), Cy2, Cy3, Cy3.5, Cy5, Cy5.5,
Cy7, Hoechst 33342, DAPI, Hoechst 33258, SYTOX Blue, chromomycin
A3, mithramycin, YOYO-1, SYTOX Orange, ethidium bromide, 7-AAD,
acridine orange, TOTO-1, TO-PRO-1, thiazole orange, TOTO-3,
TO-PRO-3, thiazole orange, propidium iodide (PI), LDS 751, Indo-1,
Fluo-3, DCFH, DHR, SNARF, Y66F, Y66H, EBFP, GFPuv, ECFP, GFP,
AmCyanl, Y77W, S65A, S65C, S65L, S65T, ZsGreenl, ZsYellowl, DsRed2,
DsRed monomer, AsRed2, mRFP1, HcRedl, monochlorobimane, calcein,
the DyLight Fluors, cyanine, hydroxycoumarin, am inocoumarin,
methoxycoumarin, Cascade Blue, Lucifer Yellow, NBD, PE-Cy5
conjugates, PE-Cy7 conjugates, APC-Cy7 conjugates, Red 613,
fluorescein, FluorX, BODIDY-FL, TRITC, Xrhodamine, Lissamine
Rhodamine B, Texas Red, TruRed, and derivatives thereof.
The term "nonexpanded" as used herein refers to a cell population
that has not been grown in culture (in vitro) to increase the
number of cells in the cell population.
The term "endogenous" as used herein refers to that which is
naturally occurring, incorporated within, housed within, adherent
to, attached to or resident in.
The term "extracellular matrix" as used herein refers to a scaffold
in a cell's external environment with which the cell interacts via
specific cell surface receptors. The extracellular matrix serves
many functions, including, but not limited to, providing support
and anchorage for cells, segregating one tissue from another
tissue, and regulating intracellular communication. The
extracellular matrix is composed of an interlocking mesh of fibrous
proteins and glycosaminoglycans (GAGs). Examples of fibrous
proteins found in the extracellular matrix include collagen,
elastin, fibronectin, and laminin. Examples of GAGs found in the
extracellular matrix include proteoglycans (e.g., heparin sulfate),
chondroitin sulfate, keratin sulfate, and non-proteoglycan
polysaccharide (e.g., hyaluronic acid). The term "proteoglycan"
refers to a group of glycoproteins that contain a core protein to
which is attached one or more glycosam inoglycans.
The term "factors" as used herein refers to nonliving components
that have a chemical or physical effect. For example, a "paracrine
factor" is a diffusible signaling molecule that is secreted from
one cell type that acts on another cell type in a tissue. A
"transcription factor" is a protein that binds to specific DNA
sequences and thereby controls the transfer of genetic information
from DNA to mRNA.
The term "fluorescence" as used herein refers to the result of a
three-state process that occurs in certain molecules, generally
referred to as "fluorophores" or "fluorescent dyes," when a
molecule or nanostructure relaxes to its ground state after being
electrically excited. Stage 1 involves the excitation of a
fluorophore through the absorption of light energy; Stage 2
involves a transient excited lifetime with some loss of energy; and
Stage 3 involves the return of the fluorophore to its ground state
accompanied by the emission of light.
The term "fluorescent-activated cell sorting" (also referred to as
"FACS") as used herein refers to a method for sorting a
heterogeneous mixture of biological cells into one or more
containers, one cell at a time, based upon the specific light
scattering and fluorescent characteristics of each cell.
The term "fossa" as used herein means a small cavity or depression,
as in a bone.
The term "fragment" as used herein refers to a small part, which
may be, without exclusion, a particle, chip, or fiber, derived
from, cut off, or broken from a larger unit which retains the
desired biological activity of the larger unit.
The term "functional equivalent" or "functionally equivalent" are
used interchangeably herein to refer to substances, molecules,
polynucleotides, proteins, peptides, or polypeptides having similar
or identical effects or use.
The term "graft" as used herein refers to a tissue or organ
transplanted from a donor to a recipient. It includes, but is not
limited to, a self-tissue transferred from one body site to another
in the same individual ("autologous graft"), a tissue transferred
between genetically identical individuals or sufficiently
immunologically compatible to allow tissue transplant ("syngeneic
graft"), a tissue transferred between genetically different members
of the same species ("allogeneic graft" or "allograft"), and a
tissue transferred between different species ("xenograft").
The term "growth" as used herein refers to a process of becoming
larger, longer or more numerous, or an increase in size, number, or
volume.
The term "growth conduction" as used herein refers to a process by
which a tissue is directed to regenerate or grow so as to conform
to a material's surface. A growth-conductive surface is one that
permits tissue growth on its surface or down into pores, channels
or pipes. Growth-conductive material facilitates the spontaneous
formation of a tissue by furnishing a microenvironment that
supports deposition or adhesion of tissuegenic cells and
optionally, vascularization. Examples of growth-conductive
materials, include, but are not limited to, processed human bone
(e.g., allograft bone, which may be an osteoconductive material),
purified collagen, calcium phosphate ceramics, synthetic polymers,
tissue-derived matrices, BMP-2 and 4, VEGF, bFGF, TGF-.beta., and
PDGF.
The term "growth-conductive matrix" as used herein refers to a
matrix that may be inert in and of itself but which supports
three-dimensional tissue formation. For example, allograft bone
tissue may be an osteoconductive matrix.
The term "growth factor" as used herein refers to extracellular
polypeptide molecules that bind to a cell-surface receptor
triggering an intracellular signaling pathway, leading to
proliferation, differentiation, or other cellular response. Growth
factors include, but are not limited to, cytokines and
hormones.
The term "growth induction" as used herein refers to a process by
which primitive, undifferentiated and tissuegenic cells are
stimulated to develop into an ensemble of cells, not necessarily
identical, that together carry out a specific function. This
ensemble of cells is termed a tissue.
The term "growth-inductive matrix" as used herein refers to a
matrix containing a substance or substances capable of recruiting
or stimulating local tissuegenic cells so that the cells are
induced (meaning to cause, bring about, bring about, or trigger) to
differentiate and/or produce a tissue.
The terms "growth-inductive components" or "growth-inductive
factors" or "tissuegenic factors" are used interchangeably to refer
to the plethora of mediators associated with tissue development and
repair.
For example, Table 11 lists exemplary growth-inductive factors
secreted by adipose tissue classified according to metabolic,
immunological or other function. (Halberg et. al., 2008,
Endocrinol. Metab. Clin. North Am., 37(3): 753-767). The
subcutaneous adipose secretome includes adiponectin, leptin, IL-6,
IL-7, IL-8, MCP-1, GRO, angiogenin, HGF, VEGF, TIMP-1, TIMP-2, etc.
(Klimkakova et. al., 2007, Biochem. Biophys. Res. Commun., 358:
897-902).
TABLE-US-00011 TABLE 11 Secreted Soluble non-ECM Factors of Adipose
Secretome Metabolic Factors Immunological Factors Other Factors
Adipsin Alpha 1 acid Angiogenin glycoprotein Adiponectin Colony
stimulating Angiopoietin 1 factor-1 Apelin Complement component
Angiopoietin 2 inhibitor C1 ApoE Complement C1 Angiotensinogen
Cortisol Complement C2 Calcitonin Insulin-like growth Complement C3
Chemerin factor 1 (IGF-1) Insulin-like growth Complement C4
Cyclophilin A factor (IGF) Binding protein 7 Complement C7
Extracellular SOD (Bp 7) Lipoprotein lipase Complement factor B
Galectin 1 Leptin Complement factor C Growth related oncogene (GRO)
Fasting induced Complement factor D Fibroblast growth factor
adipose factor (FGF) Plasminogen C reactive protein Hepatic growth
factor activated (GF) inhibitor -1 Resistin Haptoglobin
Mineralcorticoid releasing factor (MRF) Retinol binding Interleukin
1 beta Monocyte chemo- protein 4 (IL-1.beta.) attractant protein 1
(MCP-1) Vaspin Interleukin 4 (IL-4) Nerve growth factor (NGF)
Vistafin Interleukin 6 (IL-6) Pigment epithelium derived factor
(PEDF) Interleukin 7 (IL-7) Prostaglandin E2 Interleukin 8 (IL-8
Prostaglandin I2 Interleukin 10 (IL-10) Prostaglandin 2alpha
Interleukin 12 (IL-12) Serum transferring Interleukin 18 (IL-18)
Stromal derived factor 1 Lipocalin 24p3 TGF beta Macrophage
migration TIMP-1 inhibitory factor 1 Serum amyloid A3 TIMP-2 (SAA3)
Tumor necrosis factor Tissue factor alpha (TNF-.alpha.) Vascular
endothelial growth factor (VEGF)
The term "hematopoietic stem cell" refers to a cell isolated from
the blood or from the bone marrow that can renew itself,
differentiate to a variety of specialized cells, mobilize out of
the bone marrow into the circulating blood, and undergo programmed
cell death (apoptosis). According to some embodiments of the
described invention, hematopoietic stem cells derived from human
subjects express at least one type of cell surface marker,
including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59,
Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.
"HLA-DR" refers to a human class II histocompatibility antigen
present on several cell types, including antigen-presenting cells,
B cells, monocytes, macrophages, and activated T cells.
The term "interleukin" as used herein refers to a cytokine secreted
by white blood cells as a means of communication with other white
blood cells.
The term "implant" refers to any device or material inserted or
placed, permanently or temporarily, into or onto a subject as well
as those used for the administration or delivery of a therapeutic
agent(s) or substance.
The term "improve" (or improving) as used herein refers to bring
into a more desirable or excellent condition.
The terms "in the body", "void volume", "resection pocket",
"excavation", "injection site", "deposition site" or "implant site"
as used herein are meant to include all tissues of the body without
limit, and may refer to spaces formed therein from injections,
surgical incisions, tumor or tissue removal, tissue injuries,
abscess formation, or any other similar cavity, space, or pocket
formed thus by action of clinical assessment, treatment or
physiologic response to disease or pathology as non-limiting
examples thereof.
The term "indicator" as used herein refers to any substance, number
or ratio derived from a series of observed facts that may reveal
relative changes as a function of time; or a signal, sign, mark,
note or symptom that is visible or evidence of the existence or
presence thereof.
The term "inflammation" as used herein refers to the physiologic
process by which vascularized tissues respond to injury. See, e.g.,
FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed.
Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053,
incorporated herein by reference. During the inflammatory process,
cells involved in detoxification and repair are mobilized to the
compromised site by inflammatory mediators. Inflammation is often
characterized by a strong infiltration of leukocytes at the site of
inflammation, particularly neutrophils (polymorphonuclear cells).
These cells promote tissue damage by releasing toxic substances at
the vascular wall or in uninjured tissue. Traditionally,
inflammation has been divided into acute and chronic responses.
The term "acute inflammation" as used herein refers to the rapid,
short-lived (minutes to days), relatively uniform response to acute
injury characterized by accumulations of fluid, plasma proteins,
and neutrophilic leukocytes. Examples of injurious agents that
cause acute inflammation include, but are not limited to, pathogens
(e.g., bacteria, viruses, parasites), foreign bodies from exogenous
(e.g. asbestos) or endogenous (e.g., urate crystals, immune
complexes), sources, and physical (e.g., burns) or chemical (e.g.,
caustics) agents.
The term "chronic inflammation" as used herein refers to
inflammation that is of longer duration and which has a vague and
indefinite termination. Chronic inflammation takes over when acute
inflammation persists, either through incomplete clearance of the
initial inflammatory agent or as a result of multiple acute events
occurring in the same location. Chronic inflammation, which
includes the influx of lymphocytes and macrophages and fibroblast
growth, may result in tissue scarring at sites of prolonged or
repeated inflammatory activity.
The term "injury," as used herein, refers to damage or harm to a
structure or function of the body caused by an outside agent or
force, which may be physical or chemical.
The term "isolate" and its various grammatical forms as used herein
refers to placing, setting apart, or obtaining a protein, molecule,
substance, nucleic acid, peptide, cell or particle, in a form
essentially free from contaminants or other materials with which it
is commonly associated, separate from its natural environment.
The term "labeling" as used herein refers to a process of
distinguishing a compound, structure, protein, peptide, antibody,
cell or cell component by introducing a traceable constituent.
Common traceable constituents include, but are not limited to, a
fluorescent antibody, a fluorophore, a dye or a fluorescent dye, a
stain or a fluorescent stain, a marker, a fluorescent marker, a
chemical stain, a differential stain, a differential label, and a
radioisotope.
The term "labile" as used herein refers to subject to increased
degradation.
The terms "marker" or "cell surface marker" are used
interchangeably herein to refer to an antigenic determinant or
epitope found on the surface of a specific type of cell. Cell
surface markers can facilitate the characterization of a cell type,
its identification, and eventually its isolation. Cell sorting
techniques are based on cellular biomarkers where a cell surface
marker(s) may be used for either positive selection or negative
selection, i.e., for inclusion or exclusion, from a cell
population.
The term "matrix" refers to a surrounding substance within which
something is contained or embedded.
The term "mechanical agitation" as used herein refers to a process
whereby tissue is physically shaken or churned via mechanical
means. Such mechanical means include, but are not limited to, a
mixer or other mechanical device.
The term "mesenchymal stem cells (MSCs)" as used herein refers to
non-blood adult stem cells found in a variety of tissues. They are
characterized by their spindle-shape morphologically; by the
expression of specific markers on their cell surface; and by their
ability under appropriate conditions, to differentiate along a
minimum of three lineages (osteogenic, chondrogenic and
adipogenic). When referring to bone or cartilage, MSCs commonly are
known as osteochondrogenic, osteogenic, or chondrogenic, since a
single MSC has shown the ability to differentiate into chondrocytes
or osteoblasts, depending on the medium.
MSCs secrete many biologically important molecules, including
interleukins 6, 7, 8, 11, 12, 14, and 15, M-CSF, Flt-3 ligand, SCF,
LIF, bFGF, VEGF, PIGF and MCP1 (Majumdar, et al., J. Cell Physiol.
176: 57-66 (1998), Kinnaird et al, Circulation 109: 1543-49
(2004)). In 2004, it was reported that no single marker that
definitively identifies MSCs in vivo had yet been identified, due
to the lack of consensus from diverse documentations of the MSC
phenotype. Baksh, et al., J. Cell. Mol. Med. 8(3): 301-16, 305
(2004). There is general agreement that MSCs lack typical
hematopoietic antigens, namely CD14, CD34, and CD45. (Id.; citing
Pittenger, M. F. et al., Science 284: 143-47 (1999)).
The term "mill," and its various grammatical forms, as used herein
refers to operations performed to grind, to cut, to shred, to chip,
or to pulverize a substance, or equipment for performing such
operations on a substance. The term "freezer-mill" and its various
grammatical forms, as used herein refers to mill a substance in a
frozen state, or equipment for performing such operations.
The term "mounted section thickness" as used herein, refers to the
thickness of tissue sections after histological processing.
The term "multipotent" as used herein refers to a cell capable of
giving rise to a limited number of cell types of a particular cell
line.
The term "myogenic" refers to a potential of undifferentiated
precursor cells to differentiate into a muscle forming or
myocompetent cells.
The term "Optical Disector" refers to a stereological probe for
counting or selecting objects in a tissue section. This is an
extension to the basic Disector method, which is applied to a thick
section using a series, or stack, of Disectors. Rather than using
pairs of physical sections (the basic Disector method), optical
sectioning is used by creating focal planes with a thin
depth-of-field through the section. The Optical Disector begins
with a lookup section at the top of the optical disector and ends
with a reference section at the bottom of the optical disector. The
focal plane is the current reference section. The lookup section is
immediately above the focal plane. A particle in focus at the top
of the optical disector is therefore seen in the lookup section and
not counted. A particle in focus at the bottom of the optical
disector is in the reference section and therefore not in the
lookup section, is counted. Counting frame rules are applied when
the particle first comes into focus.
The term "osteoblasts" as used herein refers to cells that arise
when osteoprogenitor cells or mesenchymal cells, which are located
near all bony surfaces and within the bone marrow, differentiate
under the influence of growth factors. Osteoblasts, which are
responsible for bone matrix synthesis, secrete a collagen rich
ground substance essential for later mineralization of
hydroxyapatite and other crystals. The collagen strands to form
osteoids (spiral fibers of bone matrix). Osteoblasts cause calcium
salts and phosphorus to precipitate from the blood, which bond with
the newly formed osteoid to mineralize the bone tissue. Once
osteoblasts become trapped in the matrix they secrete, they become
osteocytes. From least to terminally differentiated, the osteocyte
lineage is (i) colony-forming unit-fibroblast (CFU-F); (ii)
mesenchymal stem cell/marrow stromal cell (MSC); (iii) osteoblast;
and (iv) osteocyte.
The term "osteocalcin" as used herein refers to a protein
constituent of bone; circulating levels are used as a marker of
increased bone turnover.
The term "osteoclast" as used herein refers to large multinucleate
cells associated with areas of bone resorption (breakdown).
The term "osteoconduction" as used herein refers to a process by
which bone is directed so as to conform to a material's surface. An
osteoconductive surface is one that permits bone growth on its
surface or down into pores, channels or pipes. Osteoconductive
material facilitates the spontaneous formation of bone by
furnishing a microenvironment that supports the ingrowth of blood
vessels, perivascular tissue and osteoprogenitor cells into the
site where it is deposited. Examples of osteoconductive materials,
include, but not limited to, processed human bone (e.g., allograft
bone), purified collagen, calcium phosphate ceramics, synthetic
polymers, BMP-2 and 4, VEGF, bFGF, TGF-.beta., and PDGF.
The term "osteoconductive matrix" as used herein refers to a matrix
that is inert in and of itself but on which cells can climb and
grow bone.
The term "osteogenic" refers to a potential of undifferentiated
precursor cells to differentiate into bone forming or
osteocompetent cells.
The term "osteogenesis" as used herein refers to the development or
formation of new bone by bone forming or osteocompetent cells.
The term "osteoinduction" as used herein refers to a process by
which primitive, undifferentiated and pluripotent cells are
stimulated to develop into a bone forming cell lineage thereby
inducing osteogenesis. For example, the majority of bone healing in
a fracture is dependent on osteoinduction. Osteoinductive materials
can be generated by combining a porous scaffold with osteogenic
cells and/or osteoinductive components, including, but not limited
to, growth factors such as BMP-2 and 4, VEGF, bFGF, TGF-.beta., and
PDGF.
The term "osteoinductive matrix" as used herein refers to a matrix
containing a substance or substances that recruit local cells to
induce (meaning to cause, bring about, bring about, or trigger)
local cells to produce bone.
The terms "osteoinductive components" or "osteogenic factors" are
used interchangeably to refer to the plethora of mediators
associated with bone development and repair, including, but not
limited to, bone morphogenic proteins (BMPs), vascular endothelial
growth factor (VEGF), basic fibroblast growth factor (bFGF),
transforming growth factor beta (TGF.beta.), and platelet-derived
growth factor (PDGF).
The term "osteointegration" refers to an anchorage mechanism
whereby nonvital components can be incorporated reliably into
living bone and that persist under all normal conditions of
loading.
The term "particle" as used herein refers to a chip, fragment,
slice, fiber or other small constituent of a larger body (e.g.,
picoparticles, nanoparticles, microparticles, milliparticle,
centiparticle, deciparticle; fractions thereof, or, in some
instances, a larger segment or piece).
The term "piece" as used herein refers to a particle, section,
strip, chip, fragment, slice, fiber or other part, derived from,
cut off, or broken from a larger unit.
The term "peptide" is used herein to refer to two or more amino
acids joined by a peptide bond.
The term "periosteum" as used herein refers to the normal
investment of bone, consisting of a dense, fibrous outer layer, to
which muscles attach, and a more delicate, inner layer capable of
forming bone.
The term "Platelet Derived Growth Factor" (PDGF) as used herein
refers to a major mitogen for connective tissue cells and certain
other cell types. It is a dimeric molecule consisting of
disulfide-bonded, structurally similar A and B-polypeptide chains,
which combine to homo- and hetero-dimers. The PDGF isoforms exert
their cellular effects by binding to and activating two
structurally related protein tyrosine kinase receptors, the
.alpha.-receptor and the .beta.-receptor. Activation of PDGF
receptors leads to stimulation of cell growth, but also to changes
in cell shape and motility; PDGF induces reorganization of the
actin filament system and stimulates chemotaxis, i.e., a directed
cell movement toward a gradient of PDGF. In vivo, PDGF plays a role
in embryonic development and during wound healing.
The term "pluripotent" as used herein refers to the ability to
develop into multiple cells types, including all three embryonic
lineages, forming the body organs, nervous system, skin, muscle and
skeleton.
The term "progenitor cell" as used herein refers to an early
descendant of a stem cell that can only differentiate, but can no
longer renew itself. Progenitor cells mature into precursor cells
that mature into mature phenotypes. Hematopoietic progenitor cells
are referred to as colony-forming units (CFU) or colony-forming
cells (CFC). The specific lineage of a progenitor cell is indicated
by a suffix, such as, but not limited to, CFU-E (erythrocytic),
CFU-F (fibroblastic), CFU-GM (granulocytic/macrophage), and
CFU-GEMM (pluripotent hematopoietic progenitor). Osteoclasts arise
from hematopoietic cells of the monocyte/neutrophil lineage
(CFU-GM). Osteoprogenitor cells arise from mesenchymal stem cells
and are committed to an osteocyte lineage.
The term "propagate" as used herein refers to reproduce, multiply,
or to increase in number, amount or extent by any process.
The term "purification" as used herein refers to the process of
isolating or freeing from foreign, extraneous, or objectionable
elements.
The term "random" as used herein refers to unpredictable. There is
some element of chance. This is the opposite of deterministic, in
which the next number or event is knowable.
The term "reduced" or "to reduce", as used herein in all of its
grammatical forms, refers to a diminishing, a decrease in, an
attenuation or abatement of the degree, intensity, extent, size,
amount, density or number of.
The term "regeneration" or "regenerate" as used herein refers to a
process of recreation, reconstitution, renewal, revival,
restoration, differentiation and growth to form a tissue with
characteristics that conform with a natural counterpart of the
tissue.
The term "relative" as used herein refers to something having, or
standing in, some significant association to something else. The
term "relative frequency" as used herein refers to the rate of
occurrence of something having or standing in some significant
association to the rate of occurrence of something else. For
example, two cell types, X cells and Y cells occupy a given
location. There are 5 X cells and 5 Y cells in that location. The
relative frequency of cell type X is 5/10; the relative frequency
of cell type Y is 5/10 in that location. Following processing,
there are 5 X cells, but only 1 Y cell in that location. The
relative frequency of cell type X following processing is 5/6, and
the relative frequency of cell type Y following processing is 1/6
in that location.
The term "repair" as used herein as a noun refers to any
correction, reinforcement, reconditioning, remedy, making up for,
making sound, renewal, mending, patching, or the like that restores
function. When used as a verb, it means to correct, to reinforce,
to recondition, to remedy, to make up for, to make sound, to renew,
to mend, to patch or to otherwise restore function. According to
some embodiments "repair" includes full repair and partial
repair.
The term "resident," and its various grammatical forms, as used
herein refers to being present habitually, existing in or intrinsic
to or incorporated therein.
The term "rinse," and its various grammatical forms, as used herein
refers to wash, to douse with a liquid or liquids or to flow a
liquid or liquids over the material being rinsed.
The term "scaffold" as used herein refers to a structure capable of
supporting a three-dimensional tissue formation. A
three-dimensional scaffold is believed to be critical to replicate
the in vivo milieu and to allow the cells to influence their own
microenvironment. Scaffolds may serve to promote cell attachment
and migration, to deliver and retain cells and biochemical factors,
to enable diffusion of vital cell nutrients and expressed products,
and to exert certain mechanical and biological influences to modify
the behavior of the cell phase. A scaffold utilized for tissue
reconstruction has several requisites. Such a scaffold should have
a high porosity and an adequate pore size to facilitate cell
seeding and diffusion of both cells and nutrients throughout the
whole structure. Biodegradability of the scaffold is also an
essential requisite. The scaffold should be absorbed by the
surrounding tissues without the necessity of a surgical removal,
such that the rate at which degradation occurs coincides as closely
as possible with the rate of tissue formation. As cells are
fabricating their own natural matrix structure around themselves,
the scaffold provides structural integrity within the body and
eventually degrades leaving the neotissue (newly formed tissue) to
assume the mechanical load.
The term "section" when used in the context of stereology refers to
a cut through material that has effectively zero thickness compared
to the size of the particles being studied. Biologists refer to
sections as thick slices through tissue. The actual thickness of
sections can leads to the Holmes effect.
The term "side-effect" as used herein refers to a result of a
therapy in addition to, or in extension of, the desired therapeutic
effect.
The term "similar" is used interchangeably with the terms
analogous, comparable, or resembling, meaning having traits or
characteristics in common.
The term "size reduction", as used herein in all of its grammatical
forms, refers to a process by which an object, such as a tissue, is
divided or reduced in size. Such processes include, without
limitation, cutting, slicing, chopping, grinding, milling,
freezer-milling, blending, homogenizing, tearing, shredding,
fracturing, breaking, crushing, and morselizing.
A "solution" generally is considered as a homogeneous mixture of
two or more substances. It is frequently, though not necessarily, a
liquid. In a solution, the molecules of the solute (or dissolved
substance) are uniformly distributed among those of the solvent.
The term "solvent" as used herein refers to a substance capable of
dissolving another substance (termed a "solute") to form a
uniformly dispersed mixture (solution).
The term "stain" as used herein refers to a composition of a dye(s)
or pigment(s) used to make a structure, a material, a cell, a cell
component, a membrane, a granule, a nucleus, a cell surface
receptor, a peptide, a microorganism, a nucleic acid, a protein or
a tissue differentiable.
The term "Sca-1" or "stem cell antigen-1" refers to a surface
protein component in a signaling pathway that affects the
self-renewal ability of mesenchymal stem cells.
The term "stem cells" refers to undifferentiated cells having high
proliferative potential with the ability to self-renew (make more
stem cells by cell division) that can generate daughter cells that
can undergo terminal differentiation into more than one distinct
cell phenotype.
The term "stereology" as used herein refers to a method of
quantifying 2D and 3D structures using estimation methods.
The term "sterilization", as used herein in all of its grammatical
forms, is any process that renders an object (e.g., a tissue, a
container for tissue, or an implement for processing tissue)
essentially free from pathogenic organisms and/or viruses by
destroying them or otherwise inhibiting their growth or vital
activity. Such processes may include exposure of the object to one
or more, without limitation, of gamma radiation, electron beam
radiation, chemical agents (e.g., alcohol, phenol, ethylene oxide
gas, acids, bases, or peroxides), heat, or ultraviolet radiation
for sufficient duration and dosages. When sterilization is
performed on a finished tissue product in its final packaging, the
process may be referred to as "terminal sterilization".
The term "stimulate", as used herein in all of its grammatical
forms, as used herein refers to activate, provoke, or spur. The
term "stimulating agent" as used herein refers to a substance that
exerts some force or effect.
The term "subcutaneous", as used herein with reference to tissues,
refers to tissues that are beneath the dermis and not part of the
dermis (i.e, the hypodermis), and may interchangeably used with the
term "subdermal".
The phrase "subject in need thereof" as used herein refers to a
patient that (i) will be administered at least one allograft, (ii)
is receiving at least one allograft; or (iii) has received at least
one allograft, unless the context and usage of the phrase indicates
otherwise.
The term "substantially similar" as used herein means that a first
value, aspect, trait, feature, number, or amount is of at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, or at
least 95% of a second value, aspect, trait, feature, number, or
amount.
The term "surfactant", as used herein, refers to a surface-active
agent that acts to reduce surface tension, which is the elastic
like force existing in the surface of a body, e.g., a liquid, at an
interface between two liquids, or that between a liquid and a
solid, tending to minimize the area of the surface, caused by
asymmetries in the intermolecular forces between surface molecules.
Surfactants usually are organic compounds that contain both
hydrophobic groups and hydrophilic groups, i.e., are amphiphilic.
Surfactants can be anionic, cationic, nonionic, and zwitterionic.
Exemplary surfactants include, but are not limited to, Triton.RTM.,
Tween.RTM. 80, egg lecithin, vitamin E-t d-.alpha.-tocopheryl
polyethylene glycol 1000 succinate (TPGS). Exemplary surfactants
suitable for use in this invention are described in, for example,
Becher, Emulsions Theory and Practice; Robert E. Krieger
Publishing, Malabar, Fla. (1965), which is incorporated herein by
reference.
The term "symptom" as used herein refers to a sign or an indication
of disorder or disease, especially when experienced by an
individual as a change from normal function, sensation, or
appearance.
The term "therapeutic effect" as used herein refers to a
consequence of treatment, the results of which are judged to be
desirable and beneficial. A therapeutic effect may include,
directly or indirectly, the arrest, reduction, or elimination of a
disease manifestation. A therapeutic effect also may include,
directly or indirectly, the arrest reduction or elimination of the
progression of a disease manifestation.
The term "tissuegenic" as used herein refers to a potential of an
undifferentiated precursor cell to differentiate into a mature cell
type and to regenerate a tissue. Exemplary tissuegenic cells
include but are not limited to a stem cell, a progenitor cell or a
combination thereof. The term "osteogenic" refers more specifically
to cell differentiation and tissue regeneration with regard to
bone.
The term "transforming growth factor beta (TGF.beta.) signaling
pathway" is used herein to refer to the signaling pathway is
involved in many cellular processes in both the adult organism and
the developing embryo including cell growth, cell differentiation,
apoptosis, cellular homeostasis and other cellular functions.
TGF.beta. superfamily ligands bind to a type II receptor, which
recruits and phosphorylates a type I receptor. The type I receptor
then phosphorylates receptor-regulated SMADs (R-SMADs) which can
now bind the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in
the nucleus where they act as transcription factors and participate
in the regulation of target gene expression.
The term "treat" or "treating" includes abrogating, substantially
inhibiting, slowing or reversing the progression of a disease,
condition or disorder, substantially ameliorating clinical or
esthetical symptoms of a condition, substantially preventing the
appearance of clinical or esthetical symptoms of a disease,
condition, or disorder, and protecting from harmful or annoying
symptoms. Treating further refers to accomplishing one or more of
the following: (a) reducing the severity of the disorder; (b)
limiting development of symptoms characteristic of the disorder(s)
being treated; (c) limiting worsening of symptoms characteristic of
the disorder(s) being treated; (d) limiting recurrence of the
disorder(s) in patients that have previously had the disorder(s);
and (e) limiting recurrence of symptoms in patients that were
previously asymptomatic for the disorder(s).
The term "vascularization" as used herein refers to a process of
ingrowth of blood vessels and perivascular tissue within a
growth-conductive matrix to support the deposition and adhesion of
tissuegenic cells to effect tissue regeneration.
The terms "VEGF", "VEGF-1" or "vascular endothelial growth
factor-1" are used interchangeably herein to refer to a cytokine
that mediates numerous functions of endothelial cells including
proliferation, migration, invasion, survival, and permeability. The
term "VEGF-2" refers to a regulator for growth of vascular
endothelial and smooth muscle cells. VEGF-2 stimulates the growth
of human vascular endothelial cells but inhibits growth of human
aortic smooth muscle cells induced by platelet-derived growth
factor.
The term "viable" as used herein refers to having the ability to
grow, expand, or develop; capable of living.
The term "xenogeneic" as used herein refers to cells or tissues
derived from individuals of different species, including, but not
limited to, porcine, bovine, caprine, equine, canine, lapine,
feline, and/or non-human mammals, such as, but not limited to,
whale, and porpoise.
Implants Containing Decellularized Matrix
Embodiments of the present invention include implants having a
decellularized matrix, and, optionally, exogenous tissuegenic
cells, growth factors, and a carrier. According to some
embodiments, the implant is in the form of a sheet. According to
some embodiments, the implant is in particulate form. According to
some embodiments, the implant is in the form of a paste, gel, or
slurry. According to some embodiments, the implant is injectable.
According to some embodiments, the implant is in a dried,
pre-formed shape. According to some embodiments, the implant is in
a porous form. According to some embodiments, the implant is in a
fibrous form. According to some embodiments, the implant fills a
void in a tissue. According to some embodiments, the implant is in
bulking agent.
According to some embodiments, the implant is a scaffold for the
delivery of growth-inductive factors. According to some
embodiments, the implant is growth-inductive. According to some
embodiments, the implant is a scaffold for the delivery of cells.
According to some embodiments, the implant is a scaffold for the
migration cells. According to some embodiments, the implant is a
growth-conductive medium for the ingrowth of tissue.
Shaped Acellular Tissue
According to some embodiments of the present invention, shaped
decelluarized tissues (also referred to herein as "shaped acellular
tissues") include three-dimensional shaped structures formed by a
process in which acellular tissues are broken into smaller
components (e.g., by milling or homogenization), then reformed into
a three-dimensional structure that is different from the source
tissue. All soft tissue types discussed in the present application
may be used, either alone or in combination with one another.
Tissue types can be allografts, autographs or xenografts. Suitable
acellular tissues may be prepared using methods disclosed in the
present application. Exemplary tissues suitable for forming shaped
acellular tissues according to embodiments of the present invention
include delipidated, decellularized adipose tissues and
delipidated, decellularized dermal tissues.
According to some embodiments of the present invention, acellular
tissues are mechanically or chemically manipulated into a
particulate form, which can be resuspended in a liquid (e.g., water
or a buffer solution) to form a flowable mass, such as a slurry.
The flowable mass may be poured into a mold of a desired shape, in
which it may form a porous or sponge-like shaped acellular tissue.
According to some embodiments, the liquid and the acellular tissue
particles are manipulated to form a putty, which can then be molded
into a desired shape. According to some embodiments, the liquid and
the acellular tissue particles are manipulated to form a paste.
According to some embodiments, the liquid and the acellular tissue
particles are manipulated to form a gel. According to some
embodiments, the gel is formed during the process used to
delipidize and decellularize the tissue. According to some
embodiments, the acellular tissue particles are mixed with a
polymer to form a paste or a putty. According to some embodiments,
the porous or sponge-like shaped acellular tissue is formed by
drying the slurry, paste or gel. According to some embodiments, the
porous or sponge-like shaped acellular tissue is formed by
lyophilizing the slurry, paste or gel. According to some
embodiments, the porosity of the shaped acellular tissue is
controlled selecting the amount of liquid relative to the amount of
particulate acellular tissue particles. According to some
embodiments, the porous or sponge-like shaped acellular material is
a solid piece that conforms to the shape of the mold after being
dried.
According to some embodiments, the shaped acellular tissue is
formed by one or more of the processes of molding a slurry, paste,
or gel, machining a sponge-like shaped acellular tissue into a
different shape, using three-dimensional ("3-D") printing to
deposit a flowable slurry, paste, or gel into a three-dimensional
shape, laminating pieces of shaped acellular tissue, and other
technologies known for use in shaping three-dimensional objects
from soft or flowable materials. According to some embodiments,
shaped acellular materials may be provided in a lyophilized,
cryopreserved, or frozen form.
According to some embodiments of the present invention, shaped
acellular tissues may be used to surgically repair defects in a
patient. According to some embodiments, shaped acellular tissues
are used alone or after being seeded or cultured with appropriate
exogenous tissuegenic cells. According to some such embodiments,
the cells may be either autologous or allogeneic, or a mixture of
autologous and allogeneic cells. According to some embodiments, the
shaped acellular tissues are provided with substances such as
growth factors, proteins, angiogenic factors and/or
controlled-release nanotubes/nanoparticles that preferentially
secrete factors for specific processes. According to some
embodiments, the substances are added to the slurry, paste, or gel
before the shaped acellular tissue is formed. According to some
embodiments, the substances are added to the shaped acellular
tissue after it is formed.
According to some embodiments of the present invention, the
degradation profile of the shaped acellular tissue and a substance
therein cause the substance to be released at an appropriate time
for growth or healing of tissues to occur. An example of this would
be to promote the formation of vasculature necessary to supply
cells within or adjacent to a shaped acellular tissue with
nutrients. In such an example, specific factors, cells, and other
substances, if needed, may be provided at selected locations on the
shaped acellular tissue to promote angiogenesis at the desired
locations. Other factors may be included that do not promote
angiogenesis, such that vasculature is formed only where is it
desired. As a more specific example, the creation of a kidney using
a shaped acellular tissue would include such factors and cells
needed to preferentially create architecture for renal arteries,
renal veins, a ureter, and other features of a functional
kidney.
According to some embodiments of the present invention, the shaped
acellular tissues have simple shapes. According to some
embodiments, the shaped acellular tissues have complex shapes.
According to some embodiments, the shaped acellular tissues have
symmetrical shapes. According to some embodiments, the shaped
acellular tissues have asymmetrical shapes. According to some
embodiments, the shaped acellular tissues have shapes similar to
the shapes of anatomical structures. According to some embodiments,
the shaped acellular tissues have the shapes of anatomical
organs.
According to some embodiments, the shaped acellular tissues are
provided for ex vivo use. According to some embodiments, the shaped
acellular tissues are provided for in vivo use (i.e., for
implantation). According to some embodiments, the shaped acellular
tissues have porosities customized for their intended use.
According to some embodiments of the present invention, the shaped
acellular tissues have pH customized for their intended use.
According to some embodiments, the shaped acellular tissues include
cross-linked collagen. According to some embodiments of the present
invention, the shaped acellular tissues include cross-linked
non-collagen components. According to some embodiments, the shaped
acellular tissues have biological polymers that are cross-linked
with non-biological (i.e., synthetic) polymers.
According to some embodiments of the present invention, a method of
forming a shaped acellular tissue includes a step of scanning or
imaging a portion of a patient's body (e.g., a portion of patient's
face or other anatomical structure), then making shaped acellular
tissues to replace those anatomical structures. In other
embodiments, a shaped acellular tissue is made to restore the shape
of an anatomical structure. In other embodiments, a shaped
acellular tissue is made to provide a substitute for a missing
anatomical structure.
According to some embodiments of the present invention, the shaped
acellular tissue is formed, then cultured in vitro with exogenous
cells. When the cells reach a sufficient number, the shaped
acellular tissue is implanted for plastic and/or reconstructive
surgery.
According to some embodiments of the present invention, the shaped
acellular tissue is formed, then cultured in vitro with cells. When
the cells reach a sufficient number, the composition is
cryopreserved, and then reconstituted when needed for use.
According to an embodiment of the present invention, mesenchymal
stem cells are harvested from a patient in need of a nasal graft,
cultured onto a shaped acellular tissue resembling the patient's
own nasal structure. After the cells have differentiated into a
sufficient number of chondrocytes, the shaped acellular tissue can
be provided to the patient as a viable graft.
According to an embodiment of the present invention, a shaped
acellular tissue is provided in a lyophilized form. The lyophilized
shaped acellular tissue is rehydrated in the operating room, where
it may be combined with such substances as the patient's
platelet-rich plasma (PRP), autologous cells such as those obtained
from the patient's bone marrow or stromal vascular fraction (SVF)
(e.g., SVF from adipose tissue obtained by liposuction), allogeneic
cells such as those obtained from a cell bank (e.g., stem cells,
progenitor cells or other cell types available from cell banks), or
bone marrow and bone marrow components including bone marrow cells
(both autologous and allogeneic).
Embodiments of the present invention include methods of making the
various embodiments of shaped acellular tissues described above.
Embodiments of such methods will be obvious to those having
ordinary skill in the art and possession of the present
disclosure.
Decellularized Matrix
According to one aspect, the described invention provides a
decellularized matrix (also referred to herein as an "acellular
matrix") derived from a biological tissue, and suitable for
implantation into a patient. The decellularized matrix comprises
ECM from which unwanted cells and cell fragments have been removed.
According to one embodiment, the tissue is autologous to the
patient. According to another embodiment, the tissue is allogeneic
to the patient. According to yet another embodiment, the tissue is
xenogeneic to the patient.
According to one embodiment, the tissue is selected from the group
consisting of an adipose tissue, an amnion tissue, an artery
tissue, a cartilage tissue, a connective tissue, a chorion tissue,
a colon tissue, a non-calcified dental tissue, a dermal tissue, a
duodenal tissue, an endothelial tissue, an epithelial tissue, a
fascial tissue, a gastrointestinal tissue, a gingival tissue, a
growth plate tissue, an intervertebral disc tissue, an intestinal
mucosal tissue, an intestinal serosal tissue, a ligament tissue, a
liver tissue, a lung tissue, a mammary tissue, a membranous tissue,
a meniscal tissue, a muscle tissue, a nerve tissue, an ovarian
tissue, a parenchymal organ tissue, a pericardial tissue, a
periosteal tissue, a peritoneal tissue, a placental tissue, a skin
tissue, a spleen tissue, a stomach tissue, a synovial tissue, a
tendon tissue, a testes tissue, an umbilical cord tissue, a
urological tissue, a vascular tissue, a vein tissue, other
non-calcified tissues, and a combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to one embodiment, the source of the
tissue is a human donor. According to one embodiment, the human
donor is a living donor. According to another embodiment, is the
human donor is a cadaveric donor. According to yet another
embodiment, the tissue donor is the intended recipient of the
acellular matrix.
Adipose Tissue
According to some embodiments, the tissue comprises an adipose
tissue derived from an adipose-rich body region.
According to some embodiments, the adipose rich body region is
selected from the group consisting of an abdomen, a hip, a
hypodermal region of skin, an infrapatellar fat pad, a knee, a
mammary organ, a thigh, and a combination thereof.
According to some embodiments, the tissue is an adipose tissue
selected from the group consisting of a visceral adipose tissue, a
hypodermal adipose tissue and, a combination thereof. According to
some embodiments, the tissue is an adipose tissue comprising a
visceral adipose tissue. According to some embodiments, the tissue
is an adipose tissue comprising a subcutaneous adipose tissue.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue is an
adipose tissue derived from an adipose-rich body region of a human
donor. According to some embodiments, the human donor is a
cadaveric donor. According to some embodiments, the human donor is
a living donor.
According to one embodiment, the tissue is an adipose tissue
derived from an autologous adipose tissue. According to one
embodiment, the tissue is an adipose tissue comprising an adipose
tissue derived from an allogeneic adipose tissue. According to one
embodiment, the tissue is an adipose tissue comprising an adipose
tissue derived from a xenogeneic adipose tissue.
Cartilage Tissue
According to some embodiments, the tissue comprises a cartilage
tissue selected from the group consisting of a hyaline cartilage
tissue, a fibrocartilage tissue, an elastic cartilage tissue, and a
combination thereof. According to some embodiments, the tissue
comprises a hyaline cartilage tissue. According to some
embodiments, the tissue comprises a fibrocartilage cartilage
tissue. According to some embodiments, the tissue comprises an
elastic cartilage tissue.
According to some embodiments, the tissue comprises a cartilage
tissue derived from a cartilaginous organ or at least one fragment
thereof.
According to some embodiments, the cartilaginous organ is selected
from the group consisting of an articular cartilage organ, a
bronchus, a growth plate, an intervertebral disc, a larynx, a
meniscus, a nose, a trachea, and a combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue
comprises a cartilage tissue derived from a human donor. According
to some embodiments, the human donor is a cadaveric donor.
According to some embodiments, the human donor is a living
donor.
According to one embodiment, the tissue is a cartilage tissue
derived from an autologous cartilage tissue. According to one
embodiment, the tissue is a cartilage tissue derived from an
allogeneic cartilage tissue. According to one embodiment, the
tissue is a cartilage tissue derived from a xenogeneic cartilage
tissue.
Non-Calcified Dental Tissue
According to some embodiments, the tissue comprises a non-calcified
dental tissue. According to some such embodiments, the tissue
comprises a dental pulp tissue.
According to some embodiments, the tissue comprises a dental pulp
tissue derived from at least one tooth. According to some
embodiments, the tissue comprises a dental pulp tissue derived from
a plurality of teeth.
According to some embodiments, the tooth is selected from the group
consisting of a deciduous tooth, a permanent tooth, and a
combination thereof. According to some embodiments, the tooth is a
deciduous tooth. According to some embodiments, the tooth is a
permanent tooth.
According to one embodiment, the source of the tissue is a dental
pulp tissue derived from a mammalian donor. According to some
embodiments, the tissue is a dental pulp tissue derived from a
human donor. According to some embodiments, the human donor is a
cadaveric donor. According to some embodiments, the human donor is
a living donor.
According to one embodiment, the tissue is an autologous dental
pulp tissue. According to one embodiment, the tissue is an
allogeneic dental pulp tissue. According to one embodiment, the
tissue is a xenogeneic dental pulp tissue.
Epithelial Tissue
According to some embodiments, the tissue comprises an epithelial
tissue selected from the group consisting of a cutaneous epithelial
tissue, a mucous epithelial tissue, a serous epithelial tissue, and
a combination thereof. According to some embodiments, the tissue
comprises a basement membrane tissue.
According to some embodiments, the tissue comprises an epithelial
tissue derived from an epithelial organ or at least one fragment
thereof.
According to some embodiments, the epithelial tissue is selected
from the group consisting of a gastrointestinal lining, an
intestinal mucosal lining, an intestinal serosal lining, a
pericardial lining, a peritoneal lining, a pleural lining, a
reproductive lining, a respiratory lining, and a urinary
lining.
According to some embodiments, the gastrointestinal lining is
selected from the group consisting of a duodenum lining, an
esophagus lining, an ileum lining, a jejunum lining, a large
intestine lining, a mouth lining, a pharynx lining, a small
intestine lining, a stomach lining, and a combination thereof.
According to some embodiments, the epithelial organ is selected
from the group consisting of a gastrointestinal organ, a
respiratory organ, a urological organ, and a combination
thereof.
According to some embodiments, the gastrointestinal organ is
selected from the group consisting of a duodenum, an esophagus, an
ileum, a jejunum, a large intestine, a mouth, a gingiva, a small
intestine, a stomach, and a combination thereof.
According to some embodiments, the respiratory organ is selected
from the group consisting of a bronchii, a diaphragm, a heart, a
larynx, a lung, a mouth, a nose, a pharynx, a trachea, and a
combination thereof.
According to some embodiments, the urological organ is selected
from the group consisting of an adrenal gland, an epididymis, a
kidney, an ovary, a penis, a prostate gland, a seminal vesicle, a
testes, a ureter, a urethra, a urinary bladder, a vas deferens, and
a combination thereof.
According to some embodiments, the epithelial organ is selected
from the group consisting of a duodenum, an esophagus, a heart, an
ileum, a jejunum, a large intestine, a lung, a mouth, a gingiva, a
pharynx, a small intestine, a skin, a stomach, and a combination
thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue is an
epithelial tissue derived from a human donor. According to some
embodiments, the human donor is a cadaveric donor. According to
some embodiments, human donor is a living donor.
According to one embodiment, the tissue is an epithelial tissue
derived from an autologous epithelial tissue. According to one
embodiment, the tissue is an epithelial tissue derived from an
allogeneic epithelial tissue. According to one embodiment, the
tissue is an epithelial tissue derived from a xenogeneic epithelial
tissue.
Fascial Tissue
According to some embodiments, the tissue comprises a fascial
tissue selected from the group consisting of a superficial fascia,
a deep fascia, a visceral fascia, and a combination thereof. The
term "fascia" as used herein refers to a fibroareolar connective
tissue lamellae distributed throughout the body surrounding
delicate organs.
According to some embodiments, the tissue comprises a fascial
tissue derived from a fascia-rich body part or at least one
fragment thereof. According to some embodiments, the fascia-rich
body part is selected from the group consisting of an arm, a back,
an elbow, a foot, a hand, a head, a knee, a leg, a muscle, a neck,
a skin, a thigh, a toe, a wrist, and a combination thereof.
According to some embodiments, the tissue comprises fascial tissue
selected from the group consisting of a myofascia associated with a
muscle, palmar fascia associated with a palm of a hand, plantar
fascia associated with a sole of a foot, thoracolumbar fascia
associated with a back, fascii lata associated with a thigh, tensor
fascia lata associated with tendon tissue, and a combination
thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue
comprises a fascia tissue derived from a human donor. According to
some embodiments, the human donor is a cadaveric donor. According
to some embodiments, human donor is a living donor.
According to one embodiment, the tissue is a fascial tissue derived
from an autologous fascia. According to one embodiment, the tissue
is a fascial tissue derived from an allogeneic fascia. According to
one embodiment, the tissue is a fascial tissue derived from a
xenogeneic fascia.
Ligament Tissue
According to some embodiments, the tissue comprises a ligament
tissue selected from the group consisting of a capsular ligament,
an extra-capsular ligament, an intracapsular ligament, a cruciate
ligament, and a combination thereof. The term "ligament" as used
herein refers to a band or sheet of fibrous tissue connecting two
or more bones, cartilages, or other structures, or serving as
support for fasciae or muscles and a fold of peritoneum supporting
any of the abdominal viscera
According to some embodiments, the tissue comprises a ligament
tissue derived from a ligament-rich body part or at least one
fragment thereof. According to some embodiments, the ligament-rich
body part is selected from the group consisting of an arm, an
elbow, a foot, a hand, a head, a knee, a leg, a neck, a pelvis, a
phalange, a thorax, a toe, a wrist, and a combination thereof.
According to some embodiments, the tissue comprises a ligament
tissue derived from a ligament organ or a fragment thereof.
According to some embodiments, the ligament organ is selected from
the group consisting of a joint, a mouth, a patella, and a
combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue
comprises a ligament tissue derived from a human donor. According
to some embodiments, the human donor is a cadaveric donor.
According to some embodiments, the human donor is a living
donor.
According to one embodiment, the tissue is a ligament tissue
derived from an autologous ligament. According to one embodiment,
the tissue is a ligament tissue derived from an allogeneic ligament
tissue. According to one embodiment, the tissue is a ligament
tissue derived from a xenogeneic ligament tissue.
Mammary Tissue
According to some embodiments, the tissue comprises a mammary
tissue derived from a mammary organ or at least one fragment
thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue is a
mammary organ from a human donor. According to some embodiments,
the human donor is a cadaveric donor. According to some
embodiments, the human donor is a living donor.
According to one embodiment, the tissue is a mammary tissue derived
from an autologous mammary organ. According to one embodiment, the
tissue is a mammary tissue derived from an allogeneic mammary
organ. According to one embodiment, the tissue is a mammary tissue
derived from a xenogeneic mammary organ.
Muscle Tissue
According to some embodiments, the tissue comprises a muscle tissue
selected from the group consisting of a cardiac muscle tissue, a
skeletal muscle tissue, a smooth muscle tissue, and a combination
thereof.
According to some embodiments, the tissue is a muscle tissue
derived from a muscle tissue-rich organ or at least one fragment
thereof.
According to some embodiments, the muscle tissue-rich organ is
selected from the group consisting of a gastrointestinal organ, a
skeletal organ, a heart, and a combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue
comprises a muscle tissue derived from a human donor. According to
some embodiments, the human donor is a cadaveric donor. According
to some embodiments, human donor is a living donor.
According to one embodiment, the tissue comprises a muscle tissue
derived from an autologous muscle tissue. According to one
embodiment, the tissue comprises a muscle tissue derived from an
allogeneic muscle tissue. According to one embodiment, the tissue
comprises a muscle tissue derived from a xenogeneic muscle
tissue.
Nerve Tissue
According to some embodiments, the tissue comprises a nerve tissue
derived from a nerve tissue-rich organ or at least one fragment
thereof.
According to some embodiments, the nerve tissue-rich organ is
selected from the group consisting of a brain, a spinal cord, and a
combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the tissue is a
nerve tissue derived from a human donor. According to some
embodiments, the human donor is a cadaveric donor.
According to one embodiment, the tissue comprises a nerve tissue
derived from an autologous nerve tissue. According to one
embodiment, the tissue comprises a nerve tissue derived from an
allogeneic nerve tissue. According to one embodiment, the tissue
comprises a nerve tissue derived from a xenogeneic nerve
tissue.
Placental Tissue
According to some embodiments, the tissue comprises a placental
tissue selected from the group consisting of an amnion tissue, a
chorion tissue, an umbilical cord tissue, and a combination
thereof.
According to some embodiments, the tissue comprises a placental
tissue derived from an isolated placental organ or at least one
fragment thereof. According to some embodiments, the placental
organ is selected from the group consisting of an amnion, a
chorion, an umbilical cord, a placenta, and a combination
thereof.
According to one embodiment, the tissue comprises a placental
tissue derived from an autologous placental tissue. According to
one embodiment, the tissue comprises a placental tissue derived
from an allogeneic placental tissue. According to one embodiment,
the tissue comprises a placental tissue derived from a xenogeneic
placental tissue.
According to one embodiment, the tissue is an umbilical cord
derived from an autologous umbilical cord. According to one
embodiment, the tissue is an umbilical cord tissue derived from an
allogeneic umbilical cord. According to one embodiment, the tissue
is an umbilical cord tissue derived from a xenogeneic umbilical
cord.
Skin Tissue
According to some embodiments, the tissue comprises a skin tissue
selected from the group consisting of an epidermal tissue, a dermal
tissue, a basement membrane tissue, and a combination thereof.
According to one embodiment, the source of the tissue is a
mammalian donor. According to some embodiments, the skin tissue is
derived from a human donor. According to some embodiments, the
human donor is a cadaveric donor. According to some embodiments,
the human donor is a living donor.
According to one embodiment, the tissue comprises a skin tissue
derived from an autologous skin tissue. According to one
embodiment, the tissue comprises a skin tissue derived from an
allogeneic skin tissue. According to one embodiment, the tissue
comprises a skin tissue derived from a xenogeneic skin tissue.
Vascular Tissue
The term "vasculature" or "vascular tissue" as used herein refers
to the vascular network of a part of the body and its arrangement.
The vascular network comprises blood vessels, i.e. any vessel
conveying blood: arteries, arterioles, capillaries, venules, and
veins. An artery is a relatively thick-walled, muscular pulsating
vessel conveying blood away from the heart. A vein is a blood
vessel carrying blood toward the heart. Both arteries and veins
comprise three layers: the tunica intima, the tunica media and the
tunica adventitia. Veins also contain valves that prevent blood
backflow. The tunica intima, a single layer of simple squamous
endothelial cells glued by a polysaccharide intercellular matrix,
surrounded by a thin layer of subendothelial connective tissue
interlaced with a number of circularly arranged elastic bands
called the internal elastic lamina; a tunica media, comprising
circularly arranged elastic fiber, connective tissue,
polysaccharide substances, and a thick elastic band called the
external elastic lamina, and the tunica adventitia, entirely made
of connective tissue. Capillaries comprise a layer of endothelium
and connective tissue.
According to some embodiments, the tissue comprises vascular
tissue. According to some embodiments, the vascular tissue is
derived from a mammalian donor. According to some embodiments, the
vascular tissue is derived from a human donor. According to some
embodiments, the vascular tissue is derived from a cadaveric donor.
According to some embodiments, the vascular tissue is derived from
a living donor.
According to one embodiment, the tissue is a vascular tissue
derived from an autologous vascular tissue. According to one
embodiment, the tissue is a vascular tissue derived from an
allogeneic vascular tissue. According to one embodiment, the tissue
is a vascular tissue derived from a xenogeneic vascular tissue.
Matrix Seeded with Tissuegenic Cells
According to some embodiments of the present invention, the matrix
is seeded with at least one viable population of tissuegenic cells.
The at least one viable population of tissuegenic cells may be
derived from one or more of the tissue types from which the matrix
may be derived. Such tissue types are identified above with respect
to the discussion of the acellular matrix.
According to one embodiment, the at least one viable population of
tissuegenic cells is selected from the group consisting of a viable
population of pluripotent stem cells, a viable population of
mesenchymal stem cells, a viable population of tissue-derived stem
cells, and a viable population of tissue-derived progenitor
cells.
According to one embodiment, the at least one viable population of
tissuegenic cells secretes at least one growth-inductive
factor.
According to one embodiment, the at least one viable population of
tissuegenic cells can be reprogrammed to at least one viable
induced pluripotent stem cell (iPSC) population.
According to one embodiment, the at least one viable population of
tissuegenic cells is capable of differentiating into cells of at
least one embryonic lineage. According to one embodiment, the
embryonic lineage is selected from the group consisting of an
ectodermal lineage, a mesodermal lineage and an endodermal
lineage.
According to one embodiment, the at least one viable population of
tissuegenic cells is capable of regenerating a target tissue.
According to one embodiment, the target tissue is selected from the
group consisting of an adipose tissue, an amnion tissue, an artery
tissue, a bone tissue, a cartilage tissue, a chorion tissue, a
colon tissue, a dental tissue, a dermal tissue, a duodenal tissue,
an endothelial tissue, an epithelial tissue, a gastrointestinal
tissue, a gingival tissue, a growth plate tissue, an intervertebral
disc tissue, an intestinal mucosal tissue, an intestinal serosal
tissue, a kidney tissue, a ligament tissue, a liver tissue, a lung
tissue, a meniscal tissue, a muscle tissue, a nerve tissue, an
ovarian tissue, a pancreatic tissue, a parenchymal organ tissue, a
pericardial tissue, a periosteal tissue, a peritoneal tissue, a
skin tissue, a spleen tissue, a synovial tissue, a tendon tissue, a
testes tissue, a urological tissue, a vascular tissue, a vein
tissue, and a combination thereof.
According to one embodiment, the at least one viable population of
tissuegenic cells is capable of differentiating into a target
tissue cell lineage. According to one embodiment, the target tissue
cell lineage is selected from the group consisting of an adipose
cell lineage, an amnion cell lineage, an artery cell lineage, a
bone cell lineage, a cartilage cell lineage, a dental cell lineage,
a dermal cell lineage, a duodenal cell lineage, an endothelial
lineage, an epithelial cell lineage, a gastrointestinal cell
lineage, a growth plate cell lineage, an intervertebral disc cell
lineage, an intestinal mucosal cell lineage, an intestinal serosal
cell lineage, a kidney cell lineage, a ligament cell lineage, a
liver cell lineage, a lung cell lineage, a meniscal cell lineage, a
muscle cell lineage, a nerve cell lineage, an ovarian cell lineage,
a pancreatic cell lineage, a parenchymal organ cell lineage, a
pericardial cell lineage, a periosteal cell lineage, a peritoneal
cell lineage, a skin cell lineage, a spleen cell lineage, a
synovial cell lineage, a tendon cell lineage, a testes cell
lineage, a urological cell lineage, a vascular cell lineage, a vein
cell lineage, and a combination thereof.
According to one embodiment, the at least one viable population of
tissuegenic cells derived from adipose tissue differentiates along
an osteogenic lineage. According to one embodiment, the at least
one viable population of tissuegenic cells derived from adipose
tissue differentiates along an adipogenic lineage. According to one
embodiment, the at least one viable population of tissuegenic cells
derived from adipose tissue differentiates along a chondrogenic
lineage. According to one embodiment, the at least one viable
population of tissuegenic cells derived from adipose tissue
differentiates along a neurogenic lineage.
According to one embodiment, the at least one viable population of
tissuegenic cells is capable of migrating from or to the at least
one growth-conductive matrix. According to one embodiment, the at
least one viable population of tissuegenic cells comprises a viable
nonexpanded population of tissuegenic cells. According to one
embodiment, the at least one viable population of tissuegenic cells
comprises a viable expanded population of tissuegenic cells.
According to some embodiments, the at least one viable population
of tissuegenic cells adherent to and resident in the endogenous
milieu of the growth conductive matrix is immune privileged. The
term "immune privileged" as used herein refers to the
characteristic of tissuegenic cells by which there is no induction
of an immune response upon transplantation of such cells.
Frequency of Tissuegenic Cells
According to some embodiments, the at least one viable population
of tissuegenic cells comprise a relative frequency substantially
similar to the total cell population of the growth-conductive
matrix. According to some embodiments, the at least one viable
population of tissuegenic cells comprise a relative frequency
substantially higher than the total cell population of the
growth-conductive matrix.
According to some embodiments, the at least one viable population
of tissuegenic cells comprise at least about at least about 10,000
tissuegenic cells per cc of implant. According to some embodiments,
the at least one viable population of tissuegenic cells comprise at
least about at least about 20,000 tissuegenic cells per cc of
implant. According to some embodiments, the at least one viable
population of tissuegenic cells comprise at least about at least
about 30,000 tissuegenic cells per cc of implant. According to some
embodiments, the at least one viable population of tissuegenic
cells comprise at least about at least about 40,000 tissuegenic
cells per cc of implant. According to some embodiments, the at
least one viable population of tissuegenic cells comprise at least
about at least about 50,000 tissuegenic cells per cc of
implant.
Growth-Inductive Component
According to some embodiments, the matrix further comprises at
least one growth-inductive component. According to some such
embodiments, the growth-inductive component is at least one
cytokine. According to some such embodiments, the at least one
growth-inductive component comprises at least one growth factor.
According to some such embodiments, the at least one growth factor
is fibroblast growth factor-2 (FGF-2). According to some such
embodiments, the at least one growth factor is fibroblast growth
factor-5 (FGF-5). According to some such embodiments, the at least
one growth factor is insulin-like growth factor-1 (IGF-1).
According to some such embodiments, the at least one growth factor
is transdermal growth factor-beta (TGF-.beta.). According to some
such embodiments, the at least one growth factor is bone
morphogenic protein-2 (BMP-2). According to some such embodiments,
the at least one growth factor is bone morphogenic protein-7
(BMP-7). According to some such embodiments, the at least one
growth factor is platelet derived growth factor (PDGF). According
to some such embodiments, the at least one growth factor is
vascular endothelial growth factor (VEGF). According to some such
embodiments, the at least one growth factor is neural epidermal
growth-factor-like 1 (NELL-1).
According to some such embodiments, the at least one
growth-inductive component is a demineralized bone matrix (DBM).
According to some such embodiments, the DBM is demineralized
autologous bone. According to some such embodiments, the DBM is
demineralized allogeneic bone. According to some such embodiments,
the DBM is demineralized xenogenic bone. According to some such
embodiments, the DBM is derived by demineralization of cancellous
bone. According to some such embodiments the DBM is derived by
demineralization of cortical bone (i.e., demineralized cortical
bone or DCB). According to some such embodiments, DBM has a
residual mineral content of 8% or less (w/w). According to some
such embodiments, DBM has a residual mineral content of 5% or less
(w/w). According to some such embodiments, DBM has a residual
mineral content of 2% or less (w/w). According to some such
embodiments, DBM has a residual mineral content of 1% or less
(w/w). According to some such embodiments, DBM has a residual
mineral content of 0.5% or less (w/w). According to some such
embodiments, DBM consists essentially of collagen, non-collagen
proteins such as growth factors, and other nonmineral substances
found in the original bone, although not necessarily in the
original quantities. According to some such embodiments, the
demineralized bone is in the form of demineralized bone fibers
(e.g., elongated particles of DBM having minimum dimensions on the
order of one micron to hundreds of microns and a maximum dimension
on the order of one millimeter to hundreds of millimeters).
According to some embodiments, the implant further comprises at
least one cryopreservative. According to some such embodiments, the
at least one cryopreservative is a solution. According to some such
embodiments, the cryopreservative is dimethylsulfoxide (DMSO).
According to some such embodiments, the cryopreservative is basal
media solution comprising about 5% DMSO. According to some such
embodiments, the cryopreservative is basal media solution
comprising about 10% DMSO. According to some such embodiments, the
cryopreservative is basal media solution comprising about 15% DMSO.
According to some such embodiments, the cryopreservative is fetal
bovine serum comprising about 5% DMSO. According to some such
embodiments, the cryopreservative is fetal bovine serum comprising
about 10% DMSO. According to some such embodiments, the
cryopreservative is a human serum comprising about 15% DMSO.
According to some such embodiments, the cryopreservative is human
serum comprising about 5% DMSO. According to some such embodiments,
the cryopreservative is human serum comprising about 10% DMSO.
According to some such embodiments, the cryopreservative is
ethylene glycol. According to some such embodiments, the
cryopreservative is propylene glycol. According to some such
embodiments, the cryopreservative is glycerol.
Method of Fabricating an Acellular Soft Tissue-Derived Matrix
Referring to FIG. 1, according to another aspect, the described
invention provides a method of fabricating an implant, the method
comprising one or more of the steps of:
(a) isolating a sample of a soft tissue from its source;
(b) pre-processing the soft tissue;
(c) delipidizing the soft tissue;
(d) decellularizing the soft tissue;
(e) disinfecting the soft tissue;
(f) post-processing the soft tissue; and
(g) packaging the soft tissue.
In various embodiments of the disclosed method, some of the
aforesaid steps may be omitted, the order of the steps may be
varied, or additional steps may be provided. The "soft tissue" of
the method may be the soft tissue in the form isolated from the
source, the pre-processed soft tissue, the delipidized soft tissue,
the decellularized (or acellular) soft tissue, the disinfected soft
tissue, the post-processed soft tissue, or the packaged soft
tissue. Embodiments of the disclosed method are discussed further
hereinbelow, and exemplary embodiments are presented.
1. Isolating Step (a): Isolating a Sample of a Soft Tissue from its
Source.
According to some embodiments, isolating step (a) comprises
excising the sample of desired soft tissue from its source.
According to some embodiments, isolating step (a) comprises
removing the sample of desired soft tissue from its source.
According to some embodiments, isolating step (a) comprises
aspirating the sample of desired soft tissue from its source.
According to some embodiments, isolating step (a) comprises
recovering the sample of desired source tissue from its source.
According to some embodiments, isolating step (a) comprises
separating the sample of desired soft tissue from adjacent tissues
of a different type than the desired soft tissue. According to some
embodiments, isolating step (a) comprises cutting the adjacent
tissues from the sample of desired soft tissue. According to some
embodiments, isolating step (a) comprises pulling the adjacent
tissues away from the sample of desired soft tissue. According to
some embodiments, isolating step (a) comprises scraping the
adjacent tissues from the sample of desired soft tissue. According
to some embodiments, isolating step (a) comprises separating the
adjacent tissues from the sample of desired soft tissue by
differential settling of the desired soft tissue and adjacent
tissues in a liquid medium.
According to some embodiments, the desired soft tissue is a
non-calcified tissue from the mammalian body. According to some
embodiments, the desired soft tissue includes one or more of an
adipose tissue, an amnion tissue, an artery tissue, a cartilage
tissue, a connective tissue, a chorion tissue, a colon tissue, a
non-calcified dental tissue, a dermal tissue, a duodenal tissue, an
endothelial tissue, an epithelial tissue, a fascial tissue, a
gastrointestinal tissue, a gingival tissue, a growth plate tissue,
an intervertebral disc tissue, an intestinal mucosal tissue, an
intestinal serosal tissue, a ligament tissue, a liver tissue, a
lung tissue, a mammary tissue, a membranous tissue, a meniscal
tissue, a muscle tissue, a nerve tissue, an ovarian tissue, a
parenchymal organ tissue, a pericardial tissue, a periosteal
tissue, a peritoneal tissue, a placental tissue, a skin tissue, a
spleen tissue, a stomach tissue, a synovial tissue, a tendon
tissue, a testes tissue, an umbilical cord tissue, a urological
tissue, a vascular tissue, a vein tissue, and other non-calcified
tissues.
According to one embodiment, the source of the desired soft tissue
is a mammalian donor. According to one embodiment, the source of
the desired soft tissue is a human donor. According to one
embodiment, the human donor is a living donor. According to another
embodiment, is the human donor is a cadaveric donor. According to
yet another embodiment, the tissue donor is the intended recipient
of the acellular matrix.
According to one embodiment, the source of the desired soft tissue
is a frozen source and the isolating step (a) includes a step of
thawing the source.
According to some embodiments, isolating step (a) is performed at a
temperature of about 25.degree. C. According to some embodiments,
isolating step (a) is performed at a temperature of about 4.degree.
C. to about 10.degree. C. According to some embodiments, isolating
step (a) is performed at an ambient temperature.
According to some embodiments, isolating step (a) comprises
reducing the bioburden of the soft tissue before it is isolated.
According to some embodiments, isolating step (a) comprises rinsing
with a liquid prior to reduce bioburden levels on the surface of
the tissue. According to some embodiments, the liquid comprises
phosphate buffered saline (PBS). According to some embodiments, the
liquid comprises acetic acid. According to some embodiments, the
liquid comprises peracetic acid.
2. Pre-Processing Step (b): Pre-Processing the Sample of Soft
Tissue.
According to some embodiments, pre-processing step (b) includes
size reduction of the sample of soft tissue. According to some
embodiments, pre-processing step (b) comprises cutting the sample
of soft tissue into strips. According to some embodiments,
pre-processing step (b) comprises slicing the sample of soft
tissue. According to some embodiments, pre-processing step (b)
comprises cutting the sample of soft tissue into chunks. According
to some embodiments, pre-processing step (b) comprises mincing the
sample of soft tissue. According to some embodiments,
pre-processing step (b) comprises grinding the sample of soft
tissue. According to some embodiments, the sample of soft tissue is
ground using a coarse plate. According to some embodiments, the
sample of soft tissue is ground using a fine plate. According to
some embodiments, separating step (b) comprises milling the sample
of desired soft tissue. According to some embodiments,
pre-processing step (b) comprises homogenizing the sample of soft
tissue. According to some embodiments, pre-processing step (b)
comprises separating components of the sample of soft tissue by
differential settling of the components of the soft tissue.
According to some embodiments, pre-processing step (b) is performed
at a temperature of about 25.degree. C. According to some
embodiments, pre-processing step (b) is performed at a temperature
of about 4.degree. C. to about 10.degree. C. According to some
embodiments, pre-processing step (b) is performed at an ambient
temperature. According to some embodiments, pre-processing step (b)
is performed at a temperature greater than an ambient temperature.
According to some embodiments, pre-processing step (b) is performed
at a physiological temperature of a living mammal. According to
some embodiments, pre-processing step (b) is performed at a
temperature of about 37.degree. C.
3. Delipidizing Step (c): Delipidizing the Sample of Soft
Tissue.
According to some embodiments, delipidizing step (c) is a step of
removing lipids from the sample of soft tissue. Some disruption of
cellular membranes and removal of cells may also occur. According
to some embodiments, delipidizing step (c) comprises removing some
of the lipids native to the soft tissue. According to some
embodiments, delipidizing step (c) comprises removing most of the
lipids native to the soft tissue. According to some embodiments,
delipidizing step (c) comprises removing substantially all of the
lipids native to the soft tissue. According to some embodiments,
delipidizing step (c) removes substantially all of the lipids
native to the soft tissue. According to some embodiments,
delipidizing step (c) disrupts cellular membranes of cells resident
in the soft tissue. According to some embodiments, delipidizing
step (c) removes cellular components from the soft tissue.
According to some embodiments, delipidizing step (c) is performed
after decellularization step (d). According to some embodiments,
multiple delipidizing steps are performed. According to some
embodiments, delipidizing step (c) is not performed.
According to some embodiments, delipidizing step (c) comprises
contacting the soft tissue with a liquid so as to separate lipids
from the soft tissue. According to some embodiments, delipidizing
step (c) comprises immersing the soft tissue in the liquid.
According to some embodiments, delipidizing step (c) comprises
soaking the soft tissue in the liquid. According to some
embodiments, delipidizing step (c) comprises mechanically agitating
the soft tissue in the liquid. According to some embodiments,
delipidizing step (c) comprises blending the soft tissue in the
liquid. According to some embodiments, blending includes a step of
mixing the soft tissue in the liquid under conditions of high
shear. According to some embodiments, blending includes a step of
mixing the soft tissue in the liquid using at least one rotating
blade rotating at a rate in the range of from about 1,000 rpm to
about 20,000 rpm. According to some embodiments, delipidizing step
(c) comprises homogenizing the soft tissue in the liquid. According
to some embodiments, homogenization includes a step of mixing the
soft tissue in the liquid such that the tissue is evenly
distributed throughout the liquid.
According to some embodiments, the liquid is water. According to
some embodiments, the liquid is an organic solvent. According to
some embodiments, the liquid is a mixture of organic solvents.
According to some embodiments, the liquid is a mixture of one or
more organic solvents and water. According to some embodiments, the
organic solvent is selected from a group consisting of a paraffin,
an aromatic hydrocarbon, a cyclic hydrocarbon, a chlorinated
hydrocarbon, a fluorinated hydrocarbon, a chlorinated methane, a
fluorinated methane, an alcohol, an ether, a ketone, an organic
acid, an aldehyde, an ester, and combinations thereof. According to
some embodiments, the organic solvent has one carbon atom.
According to some embodiments, the organic solvent has two carbon
atoms. According to some embodiments, the organic solvent has three
carbon atoms. According to some embodiments, the organic solvent
has four carbon atoms. According to some embodiments, the organic
solvent has five carbon atoms. According to some embodiments, the
organic solvent has six carbon atoms.
According to some embodiments, the liquid includes an organic acid.
According to some embodiments, the liquid includes a mineral acid.
According to some embodiments, the liquid includes an organic base.
According to some embodiments, the liquid includes a mineral base.
According to some embodiments, the liquid includes an organic salt.
According to some embodiments, the liquid contains a mineral
salt.
According to some embodiments, delipidizing step (c) comprises
recovering a lipid layer, which may be a mixture of lipid and the
aforesaid liquid, from the soft tissue. According to some
embodiments, delipidizing step (c) comprises recovering the lipid
layer by differential settling. According to some embodiments,
delipidizing step (c) comprises recovering the lipid layer by
centrifugation. According to some embodiments, delipidizing step
(c) comprises recovering the lipid layer by filtration. According
to some embodiments, delipidizing step (c) comprises recovering the
lipid layer by decantation.
According to some embodiments, delipidizing step (c) comprises
recovering the delipidized tissue. According to some embodiments,
delipidizing step (c) comprises recovering the delipidized tissue
by differential settling. According to some embodiments,
delipidizing step (c) comprises recovering the delipidized tissue
by centrifugation. According to some embodiments, delipidizing step
(c) comprises recovering the delipidized tissue by filtration.
According to some embodiments, delipidizing step (c) comprises
recovering the delipidized tissue by decantation.
According to some embodiments, delipidization step (c) comprises
contacting the soft tissue with a supercritical fluid (e.g.,
supercritical carbon dioxide). According to some embodiments,
delipidization step (c) comprises recovering the lipid by
evaporation of the supercritical fluid.
According to some embodiments, delipidization step (c) is performed
at a temperature of about 25.degree. C. According to some
embodiments, delipidization step (c) is performed at an ambient
temperature. According to some embodiments, delipidization step (c)
is performed at a temperature greater than an ambient temperature.
According to some embodiments, delipidization step (c) is performed
at a physiological temperature of a living mammal. According to
some embodiments, delipidization step (c) is performed at a
temperature of about 37.degree. C.
4. Decellularizing Step (d): Decellularizing the Sample of Soft
Tissue.
According to some embodiments, decellularization step (d) comprises
a step of removing cells and cell fragments from a sample of soft
tissue. According to some embodiments, decellularization step (d)
converts the soft tissue to an acellular matrix of ECM. According
to some embodiments, the acellular matrix is essentially free of
cell fragments. According to some embodiments, the acellular matrix
is entirely free of cell fragments. According to some embodiments,
the acellular matrix is free of native tissuegenic factors.
According to some embodiments, the acellular matrix includes native
tissuegenic factors.
According to some embodiments, decellularization step (d) comprises
contacting the sample of soft tissue with a decellularizing
solution. According to some embodiments, decellularization step (d)
comprises contacting the soft tissue with a decellularizing
solution so as to disrupt the cells and remove cells and cell
fragments from the tissue. According to some embodiments,
decellularization step (d) comprises immersing the soft tissue in
the decellularizing solution. According to some embodiments,
decellularization step (d) comprises soaking the soft tissue in the
decellularizing solution. According to some embodiments,
decellularization step (d) comprises mechanically agitating the
soft tissue in the decellularization solution. According to some
embodiments, decellularization step (d) comprises blending the soft
tissue in the decellularization solution. According to some
embodiments, blending includes a step of mixing the soft tissue in
the decellularization solution under conditions of high shear.
According to some embodiments, blending includes a step of mixing
the soft tissue with the decellularization solution using at least
one rotating blade rotating at a rate in the range of from about
1,000 rpm to about 20,000 rpm. According to some embodiments,
decellularization step (d) comprises homogenizing the soft tissue
in the decellularization solution. According to some embodiments,
homogenization includes a step of mixing the soft tissue in the
decellularization solution such that the tissue is evenly
distributed throughout the liquid. According to some embodiments,
decellularization step (d) comprises multiple decellularization
steps. According to some embodiments, decellularization step (d) is
performed before a delipidization step.
According to some embodiments, the decellularization solution is
hypertonic relative to the interior of the cells. According to some
embodiments, the decellularization solution is hypotonic relative
to the interior of the cells. According to some embodiments, the
decellularization solution includes a salt. According to some
embodiments, the decellularization solution is a salt solution.
According to some embodiments, the salt is sodium chloride.
According to some embodiments, the decellularization solution is a
pH-buffered solution. According to some embodiments, the
pH-buffered solution has a physiological pH. According to some
embodiments, the pH-buffered solution has a pH of about 7.4.
According to some embodiments, the decellularization solution
includes a detergent, emulsifier, or surfactant. According to some
embodiments, the detergent, emulsifier, or surfactant includes a
derivative of a long chain fatty acid. According to some
embodiments, the detergent, emulsifier, or surfactant includes
sodium deoxycholate. According to some embodiments, the detergent,
emulsifier, or surfactant includes sodium lauryl sulfate. According
to some embodiments, the detergent, emulsifier, or surfactant
includes sodium dodecyl sulfate. According to some embodiments, the
detergent, emulsifier, or surfactant includes a non-ionic
surfactant. According to some embodiments, the detergent,
emulsifier, or surfactant includes a polyoxyethylene derivative of
a long-chain fatty acid. According to some embodiments, the
detergent, emulsifier, or surfactant includes a polyoxyethylene
sorbitan monolaurate. According to some embodiments, the detergent,
emulsifier, or surfactant includes a polyoxyethylene derivate of an
aromatic hydrocarbon.
According to some embodiments, the concentration of the detergent,
emulsifier, or surfactant is present in a solvent in a
concentration in the range of from about 0.1% to about 5.0% (w/v).
According to some embodiments, the solvent includes water.
According to some embodiments, the solvent includes an organic
solvent. According to some embodiments, the solvent includes a
mixture of water and an organic solvent. According to some
embodiments, the solvent includes less than 20% organic solvent by
volume. According to some embodiments, the solvent includes from
about 20% about 40% organic solvent by volume. According to some
embodiments, the solvent includes from about 40% to about 60%
organic solvent by volume. According to some embodiments, the
solvent includes from about 60% to about 80% organic solvent by
volume. According to some embodiments, the solvent includes more
than 80% organic solvent by volume.
According to some embodiments, the organic solvent is selected from
a group consisting of a paraffin, an aromatic hydrocarbon, a cyclic
hydrocarbon, a chlorinated hydrocarbon, a fluorinated hydrocarbon,
a chlorinated methane, a fluorinated methane, an alcohol, an ether,
a ketone, an aldehyde, an ester, an organic acid, and combinations
thereof. According to some embodiments, the organic solvent has one
carbon atom. According to some embodiments, the organic solvent has
two carbon atoms. According to some embodiments, the organic
solvent has three carbon atoms. According to some embodiments, the
organic solvent has four carbon atoms. According to some
embodiments, the organic solvent has five carbon atoms. According
to some embodiments, the organic solvent has six carbon atoms.
According to some embodiments, the decellularization solution
includes an enzyme. According to some embodiments, the
decellularization solution includes a lipase. According to some
embodiments, the decellularization solution includes a collagenase.
According to some embodiments, the decellularization solution
includes trypsin. According to some embodiments, the
decellularization solution includes an endonuclease. According to
some embodiments, the decellularization solution includes protease.
According to some embodiments, the decellularization solution
includes a protease inhibitor.
According to some embodiments, the decellularization solution is
mildly alkaline. According to some embodiments, the
decellularization solution is mildly acidic. In some embodiments,
the decelluarization solution has a pH that is less than 6.
According to some embodiments, the decellularization solution has a
pH in the range of about 6 to about 8. In some embodiments, the
decellularization solution has a pH that is greater than 10.
According to some embodiments, the decellularization solution
includes peracetic acid.
According to some embodiments, the soft tissue is in contact with
the decellularization solution for at least 6 hours. According to
some embodiments, the soft tissue is in contact with the
decellularization solution for at least 12 hours. According to some
embodiments, the soft tissue is in contact with the
decellularization solution for at least 18 hours. According to some
embodiments, the soft tissue is in contact with the
decellularization solution for at least 24 hours. According to some
embodiments, the soft tissue is in contact with the
decellularization solution for at least 36 hours. According to some
embodiments, the soft tissue is in contact with the
decellularization solution for at least 48 hours.
According to some embodiments, decellularization step (d) comprises
a step of scraping a cellular layer from a basement membrane of the
soft tissue.
According to some embodiments, decellularization step (d) is
performed at a temperature of about 25.degree. C. According to some
embodiments, decellularization step (d) is performed at an ambient
temperature. According to some embodiments, decellularization step
(d) is performed at a temperature greater than an ambient
temperature. According to some embodiments, decellularization step
(d) is performed at a physiological temperature of a living mammal.
According to some embodiments, decellularization step (d) is
performed at a temperature of about 37.degree. C.
5. Disinfection Step (e): Disinfecting the Soft Tissue.
According to some embodiments, disinfection step (e) comprises
disrupting and/or removing micro-organisms in the soft tissue.
According to some embodiments, disinfection step (e) comprises
disrupting and/or removing viruses in the soft tissue.
According to some embodiments, disinfection step (e) comprises
contacting the soft tissue with a disinfecting solution. According
to some embodiments, disinfection step (e) comprises immersing the
soft tissue in the disinfecting solution. According to some
embodiments, disinfection step (e) comprises soaking the soft
tissue in the disinfecting solution. According to some embodiments,
disinfection step (e) comprises mechanically agitating the soft
tissue in the disinfecting solution. According to some embodiments,
disinfection step (e) comprises blending the soft tissue in the
disinfecting solution. According to some embodiments, blending
includes a step of mixing the soft tissue in the disinfecting
solution under conditions of high shear. According to some
embodiments, blending includes a step of mixing the soft tissue in
the disinfecting solution using at least one rotating blade
rotating at a rate in the range of from about 1,000 rpm to about
20,000 rpm.
According to some embodiments, the disinfecting solution includes
an antibiotic. According to some embodiments, the disinfecting
solution includes more than one antibiotic. According to some
embodiments, the disinfecting solution includes an alcohol.
According to some embodiments, the disinfecting solution includes a
glycol. According to some embodiments the disinfecting solution
includes a mixture or water with an alcohol and/or a glycol.
According to some embodiments, the disinfecting solution includes a
peroxy compound. According to some embodiments, the disinfecting
solution includes peracetic acid. According to some embodiments,
the disinfecting solution includes chlorine dioxide. According to
some embodiments, the disinfecting solution includes a detergent or
surfactant. According to some embodiments, the disinfecting
solution includes an ethylene diamine salt (e.g., ethylene diamine
tetraacetic acid (EDTA)). According to some embodiments, the
disinfecting solution includes a protein denaturant. According to
some embodiments, the disinfecting solution includes a chaotropic
salt (e.g., guanidine isothiocyanate).
According to some embodiments, the soft tissue is in contact with
the disinfecting solution for at least 1 hour. According to some
embodiments, the soft tissue is in contact with the disinfecting
solution for at least 2 hours. According to some embodiments, the
soft tissue is in contact with the disinfecting solution for at
least 4 hours.
According to some embodiments, disinfection step (e) comprises
exposing the soft tissue to ionizing radiation.
According to some embodiments, disinfection step (e) is performed
at a temperature of about 25.degree. C. According to some
embodiments, disinfection step (e) is performed at an ambient
temperature. According to some embodiments, disinfection step (e)
is performed at a temperature greater than an ambient temperature.
According to some embodiments, disinfection step (e) is performed
at a physiological temperature of a living mammal. According to
some embodiments, disinfection step (e) is performed at a
temperature of about 37.degree. C.
According to some embodiments, disinfection step (e) is performed
at a pH in the range of from about 2 to about 8, such that said
soft tissue forms a flowable gel. According to some embodiments,
disinfection step (e) is performed at a pH in the range of about 4
to about 8 such that said soft tissue forms a flowable gel.
According to some embodiments, disinfection step (e) is performed
at a pH below the isoelectric point of collagen.
According to some embodiments, the disinfected soft tissue is
washed with water to remove the disinfecting solution from the
disinfected soft tissue. According to some embodiments, the
disinfected soft tissue is washed with a buffer solution to remove
the disinfecting solution from the disinfected soft tissue.
According to some embodiments, the disinfected soft tissue is
washed with a physiological buffer to remove the disinfecting
solution from the disinfected soft tissue and bring the disinfected
tissue to a physiological pH. According to some embodiments, the
disinfected soft tissue is washed with a solution containing a
volatile polar solvent to remove the disinfecting solution from the
disinfected soft tissue.
6. Post-Processing Step (f): Post-Processing the Soft Tissue.
According to some embodiments, post-processing step (f) comprises
forming the soft tissue into a final physical form. According to
some embodiments, the final physical form of the soft tissue is a
sheet. According to some embodiments, the final physical form of
the soft tissue is particulate. According to some embodiments, the
final physical form of the soft tissue is a paste, gel, slurry, or
suspension. According to some embodiments, the final physical form
of the soft tissue is a dried, pre-formed block. According to some
embodiments, the final physical form of the soft tissue is porous.
According to some embodiments, the final physical form of the
implant is fibrous.
According to some embodiments, post-processing step (f) comprises
cutting the soft tissue to a final shape. According to some
embodiments, post-processing step (f) comprises perforating the
soft tissue. According to some embodiments, post-processing step
(f) includes air-drying the soft tissue. According to some
embodiments, post-processing step (f) includes drying the soft
tissue while heating the soft tissue. According to some
embodiments, post-processing step (f) includes lyophilizing the
soft tissue. According to some embodiments, post-processing step
(f) includes size reduction of the soft tissue. According to some
embodiments, post-processing step (f) includes milling the soft
tissue. According to some embodiments, post-processing step (f)
includes freezer-milling the soft tissue (i.e., milling the soft
tissue while it is in a frozen state, for example, by impact
milling). According to some embodiments, post-processing step (f)
includes freeze-fracturing the soft tissue. According to some
embodiments, post-processing step (f) includes compressing the soft
tissue.
According to some embodiments, post-processing step (f) comprises
rehydrating the dried soft tissue. According to some embodiments,
post-processing step (f) comprises combining particulate soft
tissue with a carrier. According to some embodiments, the carrier
includes at least one of an isotonic solution, a sodium chloride
solution, lactated Ringer's solution, a phosphate-buffered saline
solution (PBS), platelet rich plasma (PRP), hyaluronic acid (HA) or
a derivative thereof, and sodium hyaluronate. According to some
embodiments, the carrier is a sodium chloride solution at a
concentration of about 0.1% to about 1%. According to some such
embodiments, the sodium chloride solution is at a concentration of
about 0.9%. According to some embodiments, the carrier comprises
thrombin. According to some embodiments, the carrier comprises
fibrin. According to some embodiments, the carrier comprises
glycerin. According to some embodiments, the carrier comprises
collagen. According to some embodiments, the carrier comprises
lecithin. According to some embodiments, the carrier comprises a
sugar. According to some embodiments, the carrier comprises a
polysaccharide. According to some embodiments, post-processing step
(f) comprises mixing particulate soft tissue with a carrier on site
for immediate administration to a patient.
According to some embodiments, the particulate soft tissue is mixed
with a carrier such that the particulate soft tissue and carrier
combine to form a flowable gel. According to some embodiments, the
mixing step is performed at a pH in the range of from about 2 to
about 8. According to some embodiments, the mixing step is
performed at a pH in the range of about 4 to about 8. According to
some embodiments, the mixing step is performed at a pH below the
isoelectric point of collagen.
According to some embodiments, the particulate soft tissue is mixed
with a carrier such that the particulate soft tissue and the
carrier form a paste. According to some embodiments, the carrier
includes a polymer such that the particulate soft tissue and the
polymer form a paste. According to some embodiments, the polymer is
selected from the group consisting of polysaccharides, nucleic
acids, carbohydrates, proteins, polypeptides, poly(.alpha.-hydroxy
acids), poly(lactones), poly(amino acids), poly(anhydrides),
poly(orthoesters), poly(anhydride-co-imides),
poly(orthocarbonates), poly(.alpha.-hydroxy alkanoates),
poly(dioxanones), poly(phosphoesters), poly(L-lactide) (PLLA),
poly(D,L-lactide) (PDLLA), polyglycolide (PGA),
poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D, L-lactide),
poly(D,L-lactide-co-trimethylene carbonate), polyhydroxybutyrate
(PHB), poly(.epsilon.-caprolactone), poly(.delta.-valerolactone),
poly(.gamma.-butyrolactone), poly(caprolactone), polyacrylic acid,
polycarboxylic acid, poly(allylamine hydrochloride),
poly(diallyldimethylammonium chloride), poly(ethyleneimine),
polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,
polyethylene, polymethylmethacrylate, carbon fibers, poly(ethylene
glycol), poly(ethylene oxide), poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene
oxide)-co-poly(propylene oxide) block copolymers, poly(ethylene
terephthalate)polyamide, and copolymers thereof.
According to some embodiments, post-processing step (f) comprises
characterizing the acellular matrix. According to some embodiments,
post-processing step (f) comprises characterizing the acellular
matrix for DNA content, wherein absence of detectable amounts of
DNA indicates the absence of intact cells and cellular components
from the soft tissue. According to some embodiments,
post-processing step (f) comprises characterizing the acellular
matrix for the presence of growth factors. According to some
embodiments, post-processing step (f) comprises determining the
particle size distribution of particulate acellular matrix.
According to some embodiments, most of the particles have surface
areas of 625 square microns or less. According to some embodiments,
at least 80% of the particles have surface areas of 625 square
microns or less.
7. Packaging Step (g): Packaging the Soft Tissue.
According to some embodiments, packaging step (g) comprises
preparing the soft tissue for storage and subsequent use. According
to some embodiments, packaging step (g) comprises immersing the
soft tissue in a preservative solution. According to some such
embodiments, the preservative solution is aqueous ethanol.
According to some such embodiments, packaging step (g) comprises
freezing the soft tissue for storage. According to some
embodiments, packaging step (g) comprises packaging the soft tissue
in a dried or lyophilized state.
According to some embodiments, the soft tissue is in a dried or
lyophilized particulate form, and packaging step (g) comprises
packaging the dried or lyophilized tissue in a sterile container.
According to some such embodiments, packaging step (g) comprises
freezing the dried or lyophilized tissue for storage. According to
some embodiments, packaging step (g) comprises storing the dried or
lyophilized tissue at temperatures between 4.degree. C. and an
ambient temperature.
According to some embodiments, the soft tissue is packaged while in
a frozen state. According to some embodiments, the soft tissue is
provided in a frozen state, and then thawed before packaging.
According to some embodiments, the soft tissue is not in a frozen
state when provided, and is packaged while in the state in which it
is provided.
8. Addition of Tissuegenic Cells
According to some embodiments, the method of fabricating an implant
further comprises adding tissuegenic cells to the decellularized
soft tissue. Implants with added tissuegenic cells are discussed
elsewhere above. Such tissuegenic cells or implants with added
tissuegenic cells may be cryopreserved.
Cryopreservation is used for the long-term preservation of various
tissues and cells. According to some embodiments, tissuegenic cells
derived from a tissue can be cryopreserved, reconstituted, and
seeded onto an isolated matrix. According to some embodiments,
tissuegenic cells derived from a tissue can be cryopreserved,
reconstituted, and seeded onto an isolated matrix to promote
tissuegenesis in vitro and in vivo.
Detrimental effects of ice crystal formation and increased solute
concentration in cryopreserved cells can be reduced by using
cryoprotective additives or chemicals that protect cells during
freezing. Commonly used cryoprotective agents include, but are not
limited to, dimethylsulfoxide (DMSO), ethylene glycol, propylene
glycol, 2-Methyl-2.4-pentanediol (MPD), sucrose, and glycerol.
Examples of cryopreservation solutions that can be used in
preserving a tissue or a matrix include, but are not limited to, a
commercially available basal media solution such as, Mesencult
(Stem Cell Technologies), or Hyclone AdvanceStem, Fetal Bovine
Serum with 5-15% DMSO, Bovine Serum Albumin with 5-15% DMSO, Human
Serum Albumin with 5-15% DMSO, Aedesta (Cell Preservation
Solutions), LiforCell (Lifeblood Medical), ethylene glycol,
propylene glycol, and glycerol.
According to some embodiments, the packaged implant can be
preserved for an extended period of time by slowly cooling the
packaged implant in the presence of a cryoprotective agent and by
storing at ultra-low temperatures. According to some such
embodiments, packaging step (g) comprises freezing the soft tissue
implant to at least a temperature of -80.degree. C. According to
some such embodiments, packaging step (g) comprises freezing the
soft tissue implant at a controlled freezing rate. According to
some such embodiments, the controlled freezing rate is a controlled
freezing rate of about 0.5.degree. C. per minute to about
10.degree. C. per minute. According to some such embodiments, the
controlled freezing rate is a controlled freezing rate of about
1.degree. C. per minute until about -100.degree. C.
9. Addition of Growth-Inductive Components
According to some embodiments, the method of fabricating an implant
further comprises supplementing the decellularized soft tissue with
at least one growth-inductive component. According to some such
embodiments, the at least one growth-inductive component includes
one or more of a demineralized cortical bone, fibroblast growth
factor-2 (FGF-2), fibroblast growth factor-5 (FGF-5), insulin-like
growth factor 1 (IGF-1), transforming growth factor beta
(TGF-.beta.), bone morphogenic protein-2 (BMP-2), bone morphogenic
protein-7 (BMP-7), platelet-derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), neural epidermal
growth-factor-like 1 (NELL-1), and a cytokine.
According to one embodiment, the at least one growth-inductive
component is tissue-derived. According to one embodiment, the at
least one growth-inductive component comprises inducible
pluripotent stem cells (iPSCs). According to one embodiment, the at
least one growth-inductive component originates from a component of
the tissue-derived growth-inductive component other than cells.
According to one embodiment, tissuegenic cells added to the
decellularized soft tissue secrete the at least one
growth-inductive component. According to one embodiment, the
growth-inductive component comprises a growth medium derived from
expanded tissuegenic cells.
According to some embodiments, the tissue is rinsed with a liquid
prior to being separated into pieces to reduce bioburden levels on
the surface of the tissue. According to some embodiments, the
liquid comprises phosphate buffered saline (PBS). According to some
embodiments, the liquid comprises acetic acid. According to some
embodiments, the liquid comprises peracetic acid.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of exemplary embodiments of the described invention,
and are not intended to limit the scope of what the inventors
regard as their invention nor are they intended to represent that
the experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperatures, etc.) but some experimental
errors and deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, molecular weight is weight
average molecular weight, temperature is in degrees centigrade
(.degree. C.), and pressure is at or near atmospheric. Although the
exemplary embodiments are adapted to specific soft tissues, it will
be recognized by those skilled in the art that the methods
described herein can be readily adapted to other soft tissues.
Adipose, dermis, fascia, muscle, pericardium, and other connective
or membranous tissues are among the soft tissues that may be
processed according to the processes of the exemplary methods, or
variations thereof.
I. Source Tissue: Adipose Tissue
Acellular matrices derived from adipose tissue may be in a bulk
form, particulate form, or other forms. Such acellular matrices may
be used as bulking agents for cosmetic or reconstructive purposes.
Some specific applications include use as a bulking agent for to
restore natural contours where native adipose tissue has been
removed, or damaged by injury or wasting, or to correct
naturally-occurring asymmetries. In such instances, the acellular
matrices may also be used to stimulate adipose regeneration and
natural integration of the regenerated tissues. The acellular
matrices may also be used to deliver precursor cells and/or
tissuegenic factors for repair or regeneration of adipose tissue or
other tissue types. The foregoing summary of uses is intended to be
representative, and not to limit the range of uses for acellular
adipose-derived matrices according to embodiments of the present
invention.
Example 1: Decellularization with Sodium Deoxycholate
Adipose tissue is recovered aseptically from a cadaveric donor. Any
muscle, fascia, or other tissues attached to the adipose tissue, if
present, are cut away. Equal portions of the adipose tissue are
measured into empty flasks and washed by agitation in deionized
water, at a ratio of about 1000 ml of water to 500 gm of tissue.
After a period of time sufficient to remove blood and loose tissue
from the adipose tissue, the water is drained from the flask, while
collecting the adipose tissue in a 212 .mu.m to 300 .mu.m sieve.
After the water is drained, the adipose tissue is returned to the
flask.
Following the washing step, the adipose tissue is soaked with
mechanical agitation in a 4% solution of sodium deoxycholate, at a
ratio of about 1000 mL deoxycholate solution to 500 gm of adipose
tissue. After a period of time sufficient to disrupt and remove the
adipocytes and other cells from the adipose tissue, the
deoxycholate solution is drained from the flask, while collecting
the adipose tissue in a 212 .mu.m to 300 .mu.m sieve. After the
deoxycholate solution is drained, the adipose tissue is returned to
the flask, and rinsed repeatedly in deionized water to remove any
residual deoxycholate from the tissue. After each rinse, the water
is drained from the flask, as described above, and the collected
adipose tissue is returned to the flask.
Following the last water rinse, the adipose tissue is soaked in a
sterilizing solution with mechanical agitation. One suitable
sterilizing solution would be 0.5% to 1.0% peracetic acid in
deionized water. One or more additional soaks in sterilizing
solution may be needed to adequately sterilize the adipose tissue.
After the final sterilizing soak, the adipose tissue is rinsed
repeatedly in deionized water to remove any traces of sterilizing
solution from the tissue.
Following the final water rinse, the adipose tissue is stored under
refrigeration or frozen, or it is subjected to further processing
steps, such as delipidization.
Example 2: Decellularization with a Hypertonic Solution
A sample of adipose tissue is measured into a flask and a
hypertonic solution (e.g., 1M NaCl) is added in a 2:1 ratio. The
mixture is agitated for at least 12 hours at an ambient
temperature. After agitation, the hypertonic solution is decanted,
and the adipose tissue is captured in a 212 .mu.m to 300 .mu.m
sieve. The recovered adipose tissue is returned to the flask, and
soaked in an 0.1% surfactant solution with agitation for at least
12 hours at ambient temperature.
Following the last water rinse, the adipose tissue is soaked in a
sterilizing solution, with agitation. One suitable sterilizing
solution would be 0.5% to 1.0% peracetic acid in a mixture of
water, ethanol, and propylene glycol. One or more additional soaks
in sterilizing solution may be needed to adequately sterilize the
adipose tissue. After the final sterilizing soak, the adipose
tissue is rinsed repeatedly in deionized water to remove any traces
of sterilizing solution from the tissue.
Following the final water rinse, the adipose tissue is stored under
refrigeration or frozen, or it is subjected to further processing
steps, such as delipidization.
Example 3: Removal of Lipids from an Adipose Tissue
A sample of adipose tissue, which may be decellularized or
untreated, is collected and a size reduction, such as grinding or
mincing, is performed. At a temperature of ambient or greater, the
adipose tissue is placed in a conical tube or beaker, and
homogenized. The homogenized tissue is then centrifuged to separate
lipid and water layers from the tissue, taking care not to lose the
floating tissue layer.
Example 4: Delipidization of an Adipose Tissue
A sample of adipose tissue, which may be decellularized or
untreated, is collected and a size reduction, such as grinding or
mincing, is performed. After size reduction, the adipose tissue may
be processed as described in Example 2, then delipidized as
follows.
The adipose tissue is equally divided among a number of conical
tubes, and an organic solvent (e.g., an alcohol) is added to the
tube. Surfactants (e.g., Triton X-100 or Tween 80) may also be
added to promote lipid removal. The alcohol/tissue mixture is
rapidly homogenized at a temperature of ambient or greater, and the
homogenized mixture is centrifuged to separate lipid, solvent,
water and tissue layers. The lipid and solvent layers are removed
from the tube, and the tissue is recovered. The solvent
homogenization step is repeated for a number of times sufficient to
remove all of the lipid from the tissue.
After the final solvent treatment, the tissue is homogenized with
water, and the water is separated from the tissue by
centrifugation. These steps may be repeated a number of times to
remove the final traces of alcohol. The homogenate may be incubated
before it is centrifuged. A final PBS wash may be performed.
Removal of the lipids from the tissue can be verified by any of a
number of well-known lipid assays.
Example 5: Fabrication of an Acellular Implant
A sample of adipose-rich soft tissue is obtained from a suitable
donor. The tissue is inspected for muscle and dermis, which is then
cut away from the adipose tissue. Size reduction is then performed
by grinding or mincing the adipose tissue, or by another suitable
method.
The adipose tissue is then delipidized by the method of Example 3,
or by the method described herein. The adipose tissue is divided
between a number of centrifuge tubes and a small amount of water is
added to each tube to wet the tissue. The tissue is warmed to a
temperature above ambient, and centrifuged to separate a lipid
layer from the tissue. A lipid layer, a water layer, an adipose
layer, and bottom pellet are formed.
The adipose layer is recovered and blended with an equal portion of
an alcohol, or alcohol with surfactants or detergents (e.g., Triton
X-100 or Tween 80), then returned to the centrifuge tube. The
mixture of adipose tissue and alcohol is centrifuged to separate
the mixture into layers of lipid, alcohol, and adipose tissue. The
adipose tissue is then recovered. The alcohol treatment may be
repeated a number of times to remove all of the lipid from the
tissue.
After the final alcohol treatment, the adipose tissue is soaked in
a hypertonic solution (e.g., 1M NaCl) with agitation for at least 6
hours at ambient temperature. The tissue and hypertonic solution
may be blended before the soaking step begins. After soaking in
hypertonic solution, the adipose tissue is recovered from the
mixture by centrifugation at a temperature above ambient, and
soaked in a dilute detergent or surfactant solution with agitation
for at least 12 hours at ambient temperature. One suitable solution
would have at least 0.1% of a surfactant in water. After soaking in
the detergent, the adipose tissue is recovered by centrifugation,
and rinsed at least twice in deionized water.
After the water rinse, the recovered adipose tissue is suspended in
a disinfecting solution, and allowed to soak with agitation. One
suitable disinfection solution would be between 0.5% and 1%
peracetic acid in a mixture of water, ethanol, and propylene
glycol. The adipose tissue is then recovered by centrifugation, and
subjected to multiple water rinses to remove any remaining
disinfecting solution.
The rinsed adipose tissue is then dried and milled to a particulate
form. Drying may be performed by lyophilization. The dried adipose
tissue may be further milled in a frozen state (impact milling, or
freezer-milling) to further reduce the particle sizes of the
tissue, and produce a flowable particulate tissue. Suitable milling
protocols are known in the art.
The particulate tissue may be packaged in a sterile container for
later use, or thawed and rehydrated immediately. In one rehydration
method, a desired amount of the thawed particulate tissue is added
to a first syringe. The desired amount of fluid (e.g., a carrier)
is added to a second syringe, and the syringes are attached to each
other using a female-to-female locking cap. The fluid is ejected
slowly into the tissue-filled syringe, while depressing the
plungers of both syringes. The entirety of the mixture of tissue
and fluid is transferred to one of the syringes, and the other is
discarded. The syringe containing the mixture is stoppered and
packaged in foil and/ora Kapak.RTM. pouch (Kapak Corporation, St.
Louis Park, Minn.).
Example 6: Fabrication of an Implant by Reseeding Adipose-Derived
Stem Cells on a Decellularized Matrix
Adipose tissue with its endogenous stem cell niche is recovered
aseptically from a cadaveric donor within 24 hours post mortem or
from living donors undergoing elective liposuction surgery. For
example, visceral fat can be excised from cadaveric donors or
obtained with consent from living donors undergoing elective
procedures, such as liposuction, from body regions rich in adipose,
for example, hip, thigh and abdomen. Subcutaneous adipose can be
procured from the hypodermis by dissecting it out from full
thickness skin excised from cadaveric donor. Adipose tissue from
infrapatellar fat pads can be dissected out during recovery of knee
en-bloc from a cadaveric donor. The adipose tissue is stored at
4.degree. C. until ready for processing. Generally, tissue
processing commences within 72 hours post-mortem. The adipose
tissue is exposed to a bioburden reducer to generate preprocessed
adipose tissue. The preprocessed adipose tissue is subjected to a
series of PBS soaks with agitation. The preprocessed agitated
adipose tissue is then chopped into small pieces approximately
0.5.times.0.5.times.0.5 cm. The chopped adipose pieces are then
subjected to a series of rinses with cold PBS. The pH of the
rinseate is at or near physiological pH at the end of the rinse.
The rinseate is divided into two batches for stem cell isolation
and decellularized matrix preparation.
Isolation of ASCs
Viable adipose-derived stem cells (ASCs) are isolated according to
established protocols (Young et al., 2011, Acta Biomaterialia, 7:
1040-1049). Briefly, following rinses with a buffered saline
solution (e.g., 0.01M PBS, pH 7.4), one batch of the rinsed tissue
is digested with a dissociation agent (e.g., collagenase) in order
to disperse the tissue while maintaining cell viability. The digest
is subjected to centrifugation to separate the stromal vascular
fraction (SVF) rich in adipose-derived stem cells from the
supernatant rich in lipid filled adipocytes and matrix. The
supernatant is aspirated and the aspirate is frozen at -80.degree.
C. until further use. The SVF pellet is resuspended in PBS washing
solution and is subjected to a series of cold PBS washes with
alternating steps of centrifugation. Following the final wash and
resuspension in PBS, the resuspended solution is subjected to
filtration to remove undigested tissue and to obtain isolated SVF
enriched with ASCs for seeding. Alternatively, ASCs can be isolated
from the other cells present in digested adipose tissue on the
basis of cell size or immunohistochemically, for example, by using
magnetic beads, affinity chromatography, fluorescence-activated
cell sorting (FACS), flow cytometry, or with a suitable device.
Additionally, the isolated ASCs express antigens, including, but
not limited to, CD73, CD90, CD29, CD44, CD105, and/or a combination
thereof. Additionally or alternatively, the isolated ASCs do not
express antigens, including, but not limited to CD33, CD34, CD45,
CD4, CD31, CD62p CD14, HLA-DR, and/or combination thereof.
Additionally, a sample is set aside in order to evaluate the
biological activity of the tissue using commercially available
methods, including, but not limited to, for example, metabolic
assays, such as involving luciferase, tetrazolium salts, e.g.,
3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide
(MTT),
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-
razolium (MTS),
2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT), and other water soluble tetrazolium salts (e.g., WST-1, -3,
-4, -5, -8, -9, -10, and -11), and dye exclusion assays such as
Tryptan Blue.
Alternatively, isolated ASCs can be cultured without
differentiation using standard culture media typically supplemented
with 5%-20% serum. For example, the ASCs are passaged at least 5
times in such medium without differentiating, while still retaining
their multiplicity. Adipose-derived stem cells can be maintained in
control medium until 80% confluent. Cells are harvested at
confluence and population doubling calculated using the formula log
N.sub.1/log N.sub.2, where N.sub.1 is the number of cells at
confluence prior to passaging and N.sub.2 is the number of cells
seeded after passaging. Cumulative population doubling is
determined in cultures maintained until passage 13 (approximately
165 days). The mean cumulative population doubling obtained from 3
donors is expressed as a function of passage number.
Confirmation of Multi-Lineage Differentiation of Adipose-Derived
Stem Cells
Adipose-derived stem cells at passage 1 can be analyzed for their
capacity to differentiate toward the adipogenic, osteogenic,
chondrogenic, and myogenic lineages. To induce differentiation, the
stem cells are cultured with specific induction media as detailed
in Table 12.
TABLE-US-00012 TABLE 12 Lineage-specific differentiation induced by
media supplementation Medium Media Serum Supplementation Control
DMEM 10% FBS None Adipogenic (AM) DMEM 10% FBS 0.5 mM isobutyl-
methylxanthine (IBMX), 1 .mu.M dexamethasone, 10 uM insulin, 200
.mu.M indomethacin, 1% antibiotic/antimycotic Osteogenic (OM) DMEM
10% FBS 0.1 .mu.M dexamethasone, 50 .mu.M ascorbate-2- phosphate,
10 mM .beta.-glycerophosphate, 1% antibiotic/antimycotic
Chondrogenic (CM) DMEM 1% FBS 6.25 .mu.g/ml insulin, 10 ng/ml
TGF.beta.1, 50 nM ascorbate-2- phosphate, 1% antibiotic/antimycotic
Myogenic (MM) DMEM 10% FBS, 0.1 .mu.M 5% HS dexamethasone, 50 .mu.M
hydrocortisone, 1% antibiotic/antimycotic
Each media has been previously described and shown to induce
multi-lineage differentiation of MSCs (Pittenger, M. et al, 1999,
Science 284: 143-147; Grigoradis, A. et al., 1988, J. Cell. Biol.,
106: 2139-2151; Cheng, S-L. et al., 1994, Endo, 134: 277-286;
Loffler, G. et al., 1987, Klin. Wochenschr., 65: 812-817; Hauner,
H. et al., 1987, J. Clin. Endocrinol. Metabol. 64: 832-835).
Differentiation is confirmed using the histological and
immunohistological assays outlined in Table 13 and compared to a
commercial source of bone marrow-derived MSCs, lineage-specific
precursors (positive controls), and human foreskin fibroblasts
(HFFs) (negative controls)). The adipose-derived stem cells are
maintained in Control Medium.
TABLE-US-00013 TABLE 13 Differentiation Markers And Assays Of
Lineage-Specific Differentiation. Lineage-specific Histologic/
Lineage Determinant Immunohistochemical Assay Adipogenic 1. Lipid
Accumulation 1. Oil Red O stain Osteogenic 1. Alkaline 1. Alkaline
phosphatase stain phosphatase activity 2. Calcified matrix 2. Von
Kossa stain production Chondrogenic 1. Sulfated 1. Alcian Blue (pH
1.0) stain proteoglycan-rich 2. Safranin O stain matrix 2. Collagen
II 3. Collagen II-specific synthesis monoclonal antibody Myogenic
1. Multi-nucleation 1. Phase contrast microscopy 2. Skeletal muscle
2. Myosin and MyoD1 specific myosin heavy chain monoclonal
antibodies and MyoD1 expression
Adipogenesis
Adipogenic differentiation can be induced by culturing the stem
cells for 2 weeks in Adipogenic Medium (AM) and assessed using an
Oil Red O stain as an indicator of intracellular lipid accumulation
(Preece, A. 1972 A Manual for Histologic Technicians, Boston,
Mass.: Little, Brown, and Co.). Prior to staining, the cells are
fixed for 60 minutes at room temperature in 4% formaldehyde/1%
calcium and washed with 70% ethanol. The cells are incubated in 2%
(w/v) Oil Red O reagent for 5 minutes at room temperature. Excess
stain is removed by washing with 70% ethanol, followed by several
changes of distilled water. The cells are counter-stained for 2
minutes with hematoxylin.
Preparation of Decellularized Adipose Matrix
Decellularized adipose matrix is obtained either using the original
rinseate or the thawed and filtered aspirate obtained during the
ASC isolation procedure. Following a series of thorough washes with
cold PBS, the washed tissue is soaked in lysis buffer with
continuous mechanical agitation. The soaked tissue then is
subjected to cell lysis to yield a decellularized tissue. The
sterile decellularized tissue can be subjected to alternate
procedures. For example, (1) it can be lyophilized and milled using
a freezer mill to yield a decellularized adipose-derived matrix
powder; (2) it can be homogenized to obtain a decellularized
adipose-derived matrix paste or slurry; (3) it can be homogenized
and lyophilized to obtain a three-dimensional adipose-derived
matrix; or (4) it can be lyophilized to obtain an adipose
decellularized tissue matrix sheet. It can be delipidized and
decellularized according to any of the methods in Examples 1
through 5.
Recellularization
Adipose-derived decellularized matrices in powder, paste/slurry,
three-dimensional or sheet form may be used to reseed isolated
ASCs. Following the filtration step for isolating ASCs, the
isolated stromal-vascular fraction (SVF) enriched with ASCs or
otherwise purified ASCs are resuspended in basal or nutrient
enriched medium. A portion of the resuspended SVF fraction or
otherwise purified ASCs are then added to a sample of an
adipose-derived decellularized matrix produced in any form (powder,
paste/slurry, three dimensional or sheet). The decellularized
adipose matrix containing the ASCs is incubated at 37.degree. C.
The incubation step is followed by static or dynamic seeding
conditions for 24 hours, which are well known in the art. The
re-cellularized adipose matrix is then subjected to a series of
cold PBS rinses to wash away unwanted non-adherent cells.
Cryopreservation and Thawing
Additionally, prior to cryopreservation, one or more
growth-inductive components optionally can be added. These include,
but are not limited to, bone morphogenic proteins (BMPs), vascular
endothelial growth factor (VEGF), basic fibroblast growth factor
(bFGF), transforming growth factor beta (TGF.beta.),
platelet-derived growth factor (PDGF), neural epidermal
growth-factor-like 1 (NELL-1), and a combination thereof. For
cryopreservation, for example, mesencult basal media is prepared
and sterile filtered. A cryoprotectant solution in basal or
nutrient rich medium is added in order to assure full coverage of
the tissue. Exemplary cryoprotectant include, but are not limited
to, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene
glycol, 2-Methyl-2.4-pentanediol (MPD), and sucrose. The samples
are packaged in cryoresistant containers. One sample as a probe
sample is cryopreserved in a laboratory cryopreservation unit. The
packaged tissue is then subjected to slow controlled rate freezing
to at least -80.degree. C. Once the program cycle is complete, the
tissue is placed in liquid nitrogen.
Prior to implantation, a cryopreserved adipose implant is thawed.
The thaw procedure warms the tissue preparing it for implantation.
The vial containing cryopreservation solution and tissue is thawed
at room temperature or alternatively warmed to 37.degree. C. to
expedite the thawing process. Alternatively, the thawing
temperature can be at a temperature in the range of about 4.degree.
C. through 50.degree. C. Alternatively, the freeze-thawing process
can be repeated. Once the cryopreservation solution is free
flowing, the cryopreservation solution is decanted from the vial
and the tissue is implanted immediately, without any rinse. Prior
to implantation, the tissue is optionally rinsed for 0-15 minutes
with the other wash solutions including but not limited to saline,
5% dextrose in lactated ringers solution, phosphate buffered
saline, and any additional isotonic solution.
The wash solution is added at room temperature or alternatively,
prior to application to the tissue, the wash solution is warmed to
a temperature not exceeding 37.degree. C.-39.degree. C. in order to
minimize any damage to the cells contained in the tissue. The wash
solution is exchanged throughout the rinse or alternatively the
tissue is stored in the wash solution at 4.degree. C. until ready
for implantation. Any remaining tissue from the surgery is not
re-frozen for future use. All remaining tissue is disposed of
appropriately after surgery.
A strainer is used to contain the tissue during the decanting
process. This allows the cryopreservation solution and rinseate to
be removed from the tissue while minimizing any possible
contamination of tissue during preparation (minimizes human
contact). Gauze is optionally used to contain the tissue during the
decant/thaw procedure.
As described in detail above, adipose-derived stem cells possess a
potential to differentiate into a wide variety of cell types,
including, but not limited to, nerve cells, astrocytes, fat cells,
chondrogenic cells, osteogenic cells, or insulin-releasing
pancreatic cells.
II. Source Tissue: Dermis
Acellular matrices derived from the dermis may have forms such as
sheets, meshes, particles, and other forms. As sheets or meshes,
they may be used to repair or provide support for other membranous
tissues. Some specific applications for sheets or meshes include
use as slings for breast reconstruction, application to wounds or
burns where skin has been destroyed or excised, application as a
patch to repair perforations, as a support for weakened tissues, or
to cover gaps in native tissues. In particulate forms, it may be
injected as a bulking agent (e.g., to restore natural contours to
damaged or wasted tissues), or applied to wounds in a paste,
slurry, or dry form to facilitate regeneration of damaged tissue.
The acellular matrices may also be used to deliver precursor cells
and/or tissuegenic factors for repair or regeneration of dermis or
other tissue types. The foregoing summary of uses is intended to be
representative, and not to limit the range of uses for acellular
dermis-derived matrices according to embodiments of the present
invention.
Example 7: Decellularization of Tissue Using a Hypertonic
Solution
A sample of skin tissue is isolated from a suitable donor and
inspected for damage (e.g., holes or tears), and distinctive
features (e.g., moles, warts, tattoos), which are removed with a
scalpel. Tissue is inspected for hair, and same is removed using
forceps. Where the processed tissue is to be used in the form of
strips or sheets, the tissue is inspected for a uniform thickness.
Otherwise, the tissue may be reduced in size, (e.g., by grinding or
mincing). The epidermis is removed before further processing of the
dermis.
A sample of dermis is measured into a flask and a hypertonic
solution (e.g., 1M NaCl) is added in a 2:1 ratio. The mixture is
mechanically agitated for at least 12 hours at an ambient
temperature. After agitation, the hypertonic solution is decanted,
and the dermis is captured in a 212 .mu.m to 300 .mu.m sieve. The
recovered dermis is returned to the flask, and soaked in an 0.1%
surfactant solution with mechanical agitation for at least 12 hours
at ambient temperature.
Following the last water rinse, the dermis is soaked in a
sterilizing solution, with mechanical agitation. One suitable
sterilizing solution would be 0.5% to 1.0% peracetic acid in a
mixture of water, ethanol, and propylene glycol. One or more
additional soaks in sterilizing solution may be needed to
adequately sterilize the adipose tissue. After the final
sterilizing soak, the dermis is rinsed repeatedly in deionized
water to remove any traces of sterilizing solution from the
tissue.
Following the final water rinse, the dermis is stored under
refrigeration or frozen, or it is subjected to further processing
steps, such as delipidization, which may be performed by the method
of Example 3, the method of Example 4, the delipidization step of
Example 5, or other methods disclosed herein.
Example 8: Particularization of a Decellularized Dermis
A sample of dermis is decellularized as in Example 7, above. The
sample is then lyophilized according to a commercially-available
process.
The lyophilized dermis is then milled to a particulate form. It may
be further milled in a frozen state (e.g., impact milling, or
freezer-milling) to further reduce the particle size of the tissue,
and produce a flowable particulate tissue. Suitable milling
protocols are known in the art.
The particulate tissue may be packaged in a sterile container for
later use, or thawed and rehydrated immediately. In one rehydration
method, a desired amount of the thawed particulate tissue is added
to a first syringe. The desired amount of fluid (e.g., a carrier)
is added to a second syringe, and the syringes are attached to each
other using a female-to-female locking cap. The fluid is ejected
slowly into the tissue-filled syringe, while depressing the
plungers of both syringes. The entirety of the mixture of tissue
and fluid is transferred to one of the syringes, and the other is
discarded. The syringe containing the mixture is stoppered and
packaged in foil and/or ora Kapak.RTM. pouch (Kapak Corporation,
St. Louis Park, Minn.).
It will be understood that the embodiments described herein are
merely exemplary and that a person of ordinary skill in the art may
make many variations and modifications without departing from the
spirit and scope of the invention. All such variations and
modifications are intended to be included within the scope of the
invention, as defined by the appended claims.
* * * * *